JP2010500028A - Antibody to IL-17A - Google Patents

Abstract

Engineered antibodies against human IL-17A and uses thereof are provided. In one embodiment, the invention provides an antibody molecule or binding fragment thereof that binds to and inhibits the activity of a binding compound, such as human IL-17A. In some embodiments, the binding molecule comprises at least one antibody light chain variable (V _L ) domain and at least one antibody heavy chain variable (V _H ) domain or a binding fragment of these domains, wherein V _{The L} domain includes at least a specific number of complementarity determining regions (CDRs) having a sequence selected from SEQ ID NOs: 11-13, and the V _H domain is at least a specific CDR having a sequence selected from SEQ ID NOs: 14-20 Including a number, where the specific number is 1, 2 or 3.

Description

  The present invention relates generally to IL-17A specific binding compounds, such as antibodies, and uses thereof. More particularly, the present invention relates to chimeric and humanized antibodies that recognize human IL-17A and modulate its activity, particularly in inflammation, autoimmunity and proliferative disorders.

  The immune system functions to protect individuals from infectious agents such as bacteria, multicellular organisms, and viruses, and from cancer. This system includes several types of lymphoid and myeloid cells such as monocytes, macrophages, dendritic cells (DC), eosinophils, T cells, B cells and neutrophils. These lymphoid and myeloid cells frequently produce signaling proteins known as cytokines. The immune response includes inflammation, ie, the accumulation of immune cells at a specific location within the body or systemic. In response to infectious or foreign substances, immune cells secrete cytokines, which in turn modulate the proliferation, development, differentiation, or migration of immune cells. An immune response may have pathological consequences if it is associated with excessive inflammation, as in, for example, an autoimmune disease (eg, Abbas et al. (Ed.) (2000) Cellular and Molecular Immunology, W B. Saunders Co., Philadelphia, PA; Davidson and Diamond (2001) New Engl. J. Med. 345: 340-350).

  Interleukin-17A (IL-17A; cytotoxic T lymphocyte associated antigen 8 (CTLA8), also known as IL-17) is a homodimeric cytokine produced by memory T cells after antigen recognition. Generation of such T cells is promoted by interleukin-23 (IL-23). McKenzie et al. (2006) Trends Immunol. 27 (1): 17-23; Langrish et al. (2005) J, Exp, Med. 201 (2): 233-40. IL-17A acts through two receptors, IL-17RA and IL-17RC, and induces the production of many molecules involved in neutrophil biology, inflammation and organ destruction. This cytokine synergizes with tissue necrosis factor (TNF) and / or interleukin 1β (IL-1β) to promote a larger pro-inflammatory environment. Antagonizing the activity of IL-17A with an antibody or antigen-binding fragment of an antibody may cause various inflammatory, immune and proliferative disorders, such as rheumatoid arthritis (RA), osteoarthritis, rheumatoid arthritis, osteoporosis, inflammation Fibrosis (eg, scleroderma, pulmonary fibrosis, and cirrhosis), gingivitis, periodontitis, or other inflammatory periodontal diseases, inflammatory bowel disorders (eg, Crohn's disease, ulcerative colitis and inflammatory) Intestinal diseases), asthma (eg allergic asthma), allergies, chronic obstructive pulmonary disease (COPD), multiple sclerosis, psoriasis, and cancer have been proposed (eg patent document 1, patent document) 2, see Patent Documents 3 and 4).

  The most significant limitation when using antibodies as therapeutic agents in vivo is the immunogenicity of the antibodies. Since most monoclonal antibodies are derived from rodents, repeated use in humans results in the generation of an immune response against therapeutic antibodies, such as human anti-mouse antibodies, ie HAMA. Such an immune response results in at least a loss of therapeutic efficacy and, at the maximum, a potential fatal anaphylactic response. Early work to reduce the immunogenicity of rodent antibodies produced chimeric antibodies in which the mouse variable region (Fv) was fused to a human constant region. Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-43. However, mice injected with a hybrid of human variable region and mouse constant region generate a strong anti-antibody response directed against the human variable region, and possessing the entire rodent Fv region in such a chimeric antibody is a patient This suggests that it may still cause undesirable immunogenicity.

  Generally, variable domain complementarity determining region (CDR) loops are believed to contain the binding site of an antibody molecule. Accordingly, grafting (ie humanization) of rodent CDR loops to the human framework has been attempted to further minimize rodent sequences. Jones et al. (1986) Nature 321: 522; Verhoeyen et al. (1988) Science 239: 1534. However, exchanging CDR loops unfortunately has not yet resulted in an antibody with the same binding properties as the original antibody. Changes in framework residues (FR), the residues involved in supporting CDR loops in humanized antibodies, are also often required to preserve antigen binding affinity. Kabat et al. (1991) J. MoI. Immunol. 147: 1709. Although the use of CDR grafting and framework residue preserving has been reported in many humanized antibody constructs, will a particular sequence lead to an antibody with the desired binding properties and possibly biological properties? Is difficult to predict. See, eg, Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86: 10029, Gorman et al. (1991) Proc. Natl. Acad. Sci. USA 88: 4181 and Hodgson (1991) Biotechnology (NY) 9: 421-5. Furthermore, most of the previous studies used different human sequences for animal light and heavy chain variable sequences, making the predictability of such studies questionable. Known antibody sequences, or more typically, antibodies with known X-ray crystal structures such as antibodies NEW and KOL are used. See, for example, Jones et al., Supra; Verhoeyen et al., Supra; and Gorman et al., Supra. Exact sequence information has been reported for several humanized constructs.

US Patent Application Publication No. 2003/0166862 WO05 / 108616 pamphlet International Publication No. 05/051422 Pamphlet WO 06/013107 Pamphlet

  There is a need for antagonists of IL-17A, such as anti-IL-17A monoclonal antibodies, for use in the treatment of human disorders such as inflammatory, autoimmune, and proliferative disorders. Such antagonists preferably exhibit low immunogenicity in human subjects and allow repeated administration without an adverse immune response.

  The present invention relates to anti-human IL-17A antibodies having one or more desirable properties including high binding affinity, neutralizing activity, good pharmacokinetics, and low antigenicity in human subjects. The invention also relates to the use of the antibodies of the invention in the treatment of diseases.

Accordingly, in one embodiment, the invention provides an antibody molecule or binding fragment thereof that binds to and inhibits the activity of a binding compound, eg, human IL-17A. In some embodiments, the binding molecule comprises at least one antibody light chain variable (V _L ) domain and at least one antibody heavy chain variable (V _H ) domain or a binding fragment of these domains, wherein V _{The L} domain includes at least a specific number of complementarity determining regions (CDRs) having a sequence selected from SEQ ID NOs: 11-13, and the V _H domain is at least a specific CDR having a sequence selected from SEQ ID NOs: 14-20 Including a number, where the specific number is 1, 2 or 3. The specific number of CDRs may be the same or different with respect to the light and heavy chain variable domains in any given binding compound. In another embodiment, the CDRs of the V _H domain are selected from SEQ ID NOs: 14, 17, and 20. In yet another embodiment, the CDRs of the _VH domain are selected from SEQ ID NOs: 14, 16, and 19. In another embodiment, the sequences of the _VL and _VH domains are the sequences of SEQ ID NOs: 5 and 6, respectively. In some embodiments, the IL-17A binding compound inhibits the activity of human IL-17A.

In another embodiment, at least one V _L domain and at least one V _H domain binding compound, or includes binding fragments of these domains, sequences where V _L domain selected from SEQ ID NO: 26 to 28 And the V _H domain comprises CDRs 1, 2 or 3 having a sequence selected from SEQ ID NOs: 29-31. In another embodiment, the sequences of the _VL and _VH domains are the sequences of SEQ ID NOs: 22 and 23, respectively. In another embodiment, the binding compound has the same CDRs as an antibody produced from a hybridoma having ATCC accession number PTA-7739 (rat 30C10, deposited as strain JL7-30C10.C3 on July 20, 2006). .

In another embodiment, at least one V _L domain and at least one V _H domain binding compound, or includes binding fragments of these domains, sequences where V _L domain selected from SEQ ID NO: 48-50 And the V _H domain comprises CDRs 1, 2 or 3 having a sequence selected from SEQ ID NOs: 51-53.

In yet another embodiment, the binding compound comprises at least one _VL domain and at least one _VH domain, or a binding fragment of these domains, wherein the _VL domain is selected from SEQ ID NOs: 34-36. CDRs 1, 2 or 3 having a sequence and the V _H domain comprises CDRs 1, 2 or 3 having a sequence selected from SEQ ID NOs: 37-39, or _VL domains are SEQ ID NOs: 56-58. And the V _H domain comprises CDRs 1, 2 or 3 having a sequence selected from SEQ ID NOs: 59-61.

In various other embodiments, each sequence having at least 95% of the present invention is SEQ ID NO: 5 and 6, 90%, 85%, 80%, 75%, or _{V L} and _{V H} with 50% sequence homology Binding compounds that bind to human IL-17A having a domain are provided. In another embodiment, a binding compound of the invention is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more when referring to each of SEQ ID NOs: 5 and 6. Includes _VL and _VH domains with many conservative amino acid substitutions. In another embodiment, the binding compound of the invention is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or when referring to the mature form of each sequence of SEQ ID NOs: 2 and 4. Antibodies with light and heavy chains with more conservative amino acid substitutions.

In one embodiment, the binding compound is selected from the group consisting of antibodies or binding fragments thereof, such as Fab, Fab ′, Fab′-SH, Fv, scFv, F (ab ′) ₂ , and diabody. It is an antibody fragment. In one embodiment, a binding compound of the invention comprises a light chain (residues 1-220) having the mature form sequence of SEQ ID NO: 2 and a heavy chain (residues 1-220) having the mature form sequence of SEQ ID NO: 4 454). In another embodiment, the binding compound is an antibody produced from an expression vector having ATCC accession number PTA-7675 (hu16C10 in plasmid pAIL17AV1 deposited on June 28, 2006).

  In one embodiment, a binding compound of the invention comprises a heavy chain constant region, such as a human constant region, such as a γ1, γ2, γ3, or γ4 human heavy chain constant region or a variant thereof. In another embodiment, the binding compound comprises a light chain constant region, such as a human light chain constant region, such as a lambda or kappa human light chain region or variants thereof.

In another embodiment, the invention relates to an isolated nucleic acid, such as DNA, encoding a binding compound of the invention, such as an antibody (or binding fragment thereof) that binds human IL-17A. In one embodiment, the isolated nucleic acid comprises at least one antibody light chain variable (V _L ) domain and at least one antibody heavy chain variable (V _H ) domain, or a binding fragment of these domains. Wherein the _VL domain comprises at least a particular number of complementarity determining regions (CDRs) having a sequence selected from SEQ ID NOs: 11-13, and the _VH domain is selected from SEQ ID NOs: 14-20 At least a specific number of CDRs having the sequence, wherein the specific number is 1, 2 or 3.

  In another embodiment, the isolated nucleic acid encodes one or both of the light and heavy chain variable region sequences of SEQ ID NOs: 5 and 6, respectively. In yet another embodiment, the isolated nucleic acid encodes antibody 16C10 comprising a light chain having the mature form of SEQ ID NO: 2 and a heavy chain having the mature form of SEQ ID NO: 4. In some embodiments, the isolated nucleic acid comprises nucleotides 58-717 of SEQ ID NO: 1 or SEQ ID NO: 62, and in other embodiments, the isolated nucleic acid is SEQ ID NO: 3 or SEQ ID NO: 63. Contains nucleotides 58-1419. In yet another embodiment, the isolated nucleic acid comprises the sequence of SEQ ID NO: 1 and the sequence of SEQ ID NO: 3. In yet another embodiment, the isolated nucleic acid comprises the sequence of SEQ ID NO: 62 and the sequence of SEQ ID NO: 63. In some embodiments, an isolated nucleic acid encodes both the light and heavy chains on a single nucleic acid molecule, and in other embodiments, the light and heavy chains are separate nucleic acid molecules 2. Coded to more than one.

  In a further embodiment, the invention relates to an expression vector comprising an isolated nucleic acid of the invention, wherein the nucleic acid is operably linked to a regulatory sequence that is recognized by the host cell when the host cell is transfected with the vector. is doing. In one embodiment, the expression vector has ATCC accession number PTA-7576 (hu16C10 in plasmid pAIL17AV1 deposited on June 28, 2006).

  In another embodiment, the present invention relates to a host cell comprising the expression vector of the present invention. The invention further relates to a method for producing a binding compound of the invention comprising culturing a host cell carrying an expression vector encoding the binding compound in a medium and isolating the binding compound from the host cell or medium.

  The invention also cross-blocks the binding of an antibody or binding fragment thereof that binds to the same epitope on human IL-17A as antibody 16C10, 4C3, 30C10, 12E6, 23E12, or 1D10, eg, any of these antibodies of the invention. Or a binding compound such as an antibody that binds within an epitope defined by amino acid residues 74-85 of human IL-17A (SEQ ID NO: 40).

The present invention also provides antibodies such as binding compounds that bind with an equilibrium dissociation constant (K _d ) (ie higher affinity) of 1000, 500, 100, 50, 20, 10, 5, 2 pM or lower. Or a high affinity human IL-17A binding molecule such as a binding fragment thereof. The invention also provides a biology in which IL-17A stimulation is performed at a concentration of 1000 pM (1 nM), such as IL-17A stimulated production of IL-6 from normal human fibroblasts, foreskin fibroblasts, or synoviocytes. Relates to binding compounds having a strong biological activity such as an IC ₅₀ of 5000, 2000, 1000, ₅₀₀ pM when measured in a clinical activity test. The present invention also provides 1000, 500, 200, 100, 50 pM when measured in a biological activity test in which IL-17A stimulation is performed at a concentration of 100 pM, such as a Ba / F3-hIL-17Rc-mGCSFR proliferation test. It relates to binding compounds having a lower IC ₅₀ than this. In general, the present invention relates to IL-17A concentrations of 10 ×, 5 ×, 2 ×, 1 × when the concentration of IL-17A is, for example, 5, 10, 50, 100, 500 or 1000 pM or higher. And binding compounds capable of inhibiting the activity of human IL-17A in biological tests at concentrations in the range of values as low as 0.5 ×.

  The present invention also provides IL-17A-induced neutrophils to the lungs up to 50% or more when administered to mice to produce serum concentrations of binding compounds of 50, 40, 30, 20 μg / ml or lower. The present invention relates to a binding compound capable of reducing recruitment.

In one embodiment, the binding compound binds to cynomolgus IL-17A with an affinity that is 5 times, 10 times, or 20 times less than its affinity for human IL-17A (K _d ). To do. In another embodiment, the binding compound binds to human IL-17A with an affinity that is 100, 500, 1000, or 2000 times greater than its affinity (K _d ) for mouse or rat IL-17A.

  The invention also relates to a method of treating a subject, including a human subject in need of treatment with a human IL-17A binding compound of the invention. Such subjects include inflammatory or autoimmune disorders such as rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis, chronic obstructive pulmonary disease, cystic fibrosis, systemic sclerosis, autografts You may have rejection, autoimmune myocarditis, or peritoneal adhesions. Such therapeutic methods further comprise administering one or more additional therapeutic agents such as immunosuppressive or anti-inflammatory agents. In one embodiment, the subject has been diagnosed with an IL-17A mediated disease. In another embodiment, the subject has been diagnosed with rheumatoid arthritis. In yet another embodiment, the subject has been diagnosed with multiple sclerosis.

  In another embodiment, the invention provides a therapeutic method comprising administration of a therapeutically effective amount of an anti-human IL-17A antibody or binding fragment in combination with one or more other therapeutic agents. In one embodiment, the other therapeutic agent is an anti-human IL-23 antibody, or a binding fragment thereof. In various embodiments, the anti-human IL-23 antibody, or binding fragment thereof, is administered before, simultaneously with, or after the anti-human IL-17A antibody or fragment. In one embodiment, anti-IL-17A and anti-IL-23 antibodies are administered together for a limited time during the acute phase of an adverse immunological event, after which treatment with anti-IL-17A antibodies is interrupted However, treatment with anti-IL-23 antibody continues. In other embodiments, the other one or more agents are IL-1β, IL-6, or TGF-β antagonists, such as anti-IL-6 or anti-TGF-β antibodies, or such antagonists. Includes combinations.

  The invention also relates to compositions and formulations of the binding compounds of the invention comprising the binding compound and a pharmaceutically acceptable carrier or diluent, and optionally one or more immunosuppressive or anti-inflammatory agents.

Figure 2 shows the alignment of light chain variable domains of several anti-human IL-17A antibodies according to the present invention. Rat 16C10V _L = SEQ ID NO: 7; Human 16C10V _L = SEQ ID NO: 5; Rat 4C3V _L = SEQ ID NO: 21; Human 4C3V _L = SEQ ID NO: 5; Rat 23E12V _L = SEQ ID NO: 45; Rat 30C10V _L = SEQ ID NO: 24; Human 30C10V _L = SEQ ID NO: 22; rat 12E6V _L = SEQ ID NO: 32; rat 1D10V _L = SEQ ID NO: 54. CDRs are shown (and presented in Table 3). Numbering is referred to herein as “Kabat et al. (1991)”, Kabat et al. (1991) “Sequences of Proteins of Immunological Interest”, U.S. Pat. S. Department of Health and Human Services, NIH Pub. 91-3242, 5th edition. 2 shows the alignment of heavy chain variable domains of several anti-human IL-17A antibodies according to the present invention. Rat 16C10V _H = SEQ ID NO: 8; human 16C10V _H = SEQ ID NO: 6; rat 4C3V _H = SEQ ID NO: 8; human 4C3V _H = SEQ ID NO: 6; rat 23E12V _H = SEQ ID NO: 47; rat 30C10V _H = SEQ ID NO: 25; human 30C10V _H = SEQ ID NO: 23; rat 12E6V _H = SEQ ID NO: 33; rat 1D10V _H = SEQ ID NO: 55. CDRs are shown (and presented in Table 4). Numbering follows Kabat et al. (1991). 2 shows the amino acid sequence of the light chain of humanized anti-IL-17A antibody 16C10 according to the present invention (mature form of SEQ ID NO: 2, ie residues 1-220). CDRs are shown. 2 shows the amino acid sequence of the heavy chain of humanized anti-IL-17A antibody 16C10 according to the present invention (mature form of SEQ ID NO: 4, ie, residues 1 to 454). CDRs are shown. Figures 3A-3D show the effect of anti-IL-17A antibody treatment on pathology in a CIA mouse model of rheumatoid arthritis. Treatment includes administration of anti-IL-17A antibody 1D10 (28, 7 and 2 mg / kg) and isotype control (7 mg / kg). FIG. 3A shows visual disease severity score (DSS), ie visual foot edema and erythema, as a function of antibody treatment. Score = 0 = foot has a similar appearance to control (untreated) foot; 1 = inflammation of one finger on a given foot; 2 = inflammation of 2 fingers or palm of a given foot; 3 = 1 given Inflammation of palms and fingers of the foot. Figures 3A-3D show the effect of anti-IL-17A antibody treatment on pathology in a CIA mouse model of rheumatoid arthritis. Treatment includes administration of anti-IL-17A antibody 1D10 (28, 7 and 2 mg / kg) and isotype control (7 mg / kg). FIG. 3B shows cartilage damage (by histopathology) as a function of antibody treatment. Score 0 = normal; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe. Figures 3A-3D show the effect of anti-IL-17A antibody treatment on pathology in a CIA mouse model of rheumatoid arthritis. Treatment includes administration of anti-IL-17A antibody 1D10 (28, 7 and 2 mg / kg) and isotype control (7 mg / kg). FIG. 3C shows bone erosion (by histopathology) as a function of antibody treatment. Score 0 = normal; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe. Figures 3A-3D show the effect of anti-IL-17A antibody treatment on pathology in a CIA mouse model of rheumatoid arthritis. Treatment includes administration of anti-IL-17A antibody 1D10 (28, 7 and 2 mg / kg) and isotype control (7 mg / kg). FIG. 3D shows bone erosion (by histopathology) on the feet of CIA mice scored 2 or 3 in the visual DSS, ie, feet with high inflammation. rIgG1 was used as an isotype control antibody. Score 0 = normal; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe. The% BAL neutrophils (pulmonary favor) in mice administered intranasally with various anti-human IL-17A antibodies of the invention (1D10, 16C10, 4C3) and human IL-17A as a function of serum concentration of isotype control. Measures for sphere recruitment). Black triangles represent control experiments where no anti-IL-17A antibody was added, and the leftmost data points (white triangles) represent unstimulated controls. 2 shows the nucleotide sequence (SEQ ID NO: 62) encoding the light chain of humanized anti-IL-17A antibody 16C10. 2 shows the nucleotide sequence (SEQ ID NO: 63) encoding the heavy chain of humanized anti-IL-17A antibody 16C10.

1. Definitions In order that the present invention may be more readily understood, certain technical terms are specifically defined below. Unless defined otherwise herein, all other technical terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

  In this specification, including the appended claims, the singular forms include the corresponding plural forms unless the context clearly dictates otherwise.

  “Activation”, “stimulation” and “treatment” when applied to a cell or receptor have similar meanings, eg, a cell or receptor with a ligand, unless otherwise indicated in context. Activation, stimulation or treatment. “Ligand” includes natural and synthetic ligands such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies. “Ligand” also encompasses small molecules, such as peptide mimetics of cytokines and peptide mimetics of antibodies. “Activation” may refer to cell activation as regulated by internal mechanisms as well as by external or environmental factors. For example, a “response” of a cell, tissue, organ, or organism is a biochemical or physiological behavior, such as concentration, density, adhesion or migration within a biological compartment, ratio of gene expression, or state of differentiation. In which case the change correlates with activation, stimulation, or treatment, or an internal mechanism such as genetic programming.

  “Activity” of a molecule describes the binding of the molecule to a ligand or receptor, catalytic activity; the ability to stimulate gene expression or cell signaling, differentiation or maturation; antigenic activity, modulation of the activation of other molecules, etc. or May point. “Activity” of a molecule can also refer to activity in modulating or maintaining cell-cell interactions, eg adhesion, or activity in maintaining the structure of a cell, eg cell membrane or cytoskeleton. “Activity” may also mean specific activity, eg, [catalytic activity] / [mg protein] or [immunological activity] / [mg protein] in a biological compartment. “Activity” may also refer to modulation of elements of the innate or adaptive immune system. "Proliferative activity" refers to activities that promote, require, or are specifically associated with, for example, normal cell division and cancer, tumors, dysplasia, cell transformation, metastasis, and angiogenesis Is included.

  “Administration” and “treatment” refer to an animal, human, subject, cell, tissue, organ, or biological fluid when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid. Refers to the contact of an exogenous pharmaceutical, therapeutic, diagnostic, or composition with. “Administration” and “treatment” may refer to methods of treatment, pharmacokinetics, diagnosis, research and experiment. Cell therapy includes contact of the reagent with the cell as well as contact of the reagent with the fluid, where the fluid is in contact with the cell. “Administration” and “treatment” also means in vitro and ex vivo treatment of cells, for example, with reagents, diagnostic agents, binding compounds, or with another cell. “Treatment” when applied to humans, livestock or research subjects refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications. “Treatment” when applied to a human, domestic animal or research subject or cell, tissue or organ refers to an IL-17A agonist or to a human or animal subject, cell, tissue, physiological compartment, or physiological fluid or Includes contact with an IL-17A antagonist. “Cell therapy” also refers to the situation where an IL-17A agonist or IL-17A antagonist contacts the IL-17A receptor, eg, in the fluid phase or colloidal phase, but further the agonist or antagonist is the cell or receptor. This includes situations where there is no contact.

  “Treating” or “treating” refers to a therapeutic agent, such as a composition containing any of the binding compounds of the invention, for one or more symptoms of the disease for which the agent has a known therapeutic activity. It is meant to be administered internally or externally to a patient having Typically, the agent induces or suppresses the progression of one or more symptoms of the disease in the treated patient or population to any clinically measurable extent. Administered in an amount effective to alleviate. The amount of therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as “therapeutically effective amount”) is the condition of the disease, the age and weight of the patient, and the drug that elicits the desired response in the patient. May vary depending on factors such as ability. Whether a disease symptom is alleviated can be assessed by any clinical instrumentation typically used by physicians or other medical professionals to assess the severity or progression of the symptom. Embodiments of the invention (eg, methods of treatment or articles of manufacture) need not be effective in alleviating target disease symptoms in any patient, but it may be any statistical test known in the art, eg Target disease in a statistically significant number of patients as measured by Student's t test, Chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test The symptoms must be reduced.

  Reference is made herein to four variants of the human IL-17A protein. i) As used herein, “human IL-17A” and “native human IL-17A” (“huIL-17A” and “humIL-17A”) are human IL-17A protein accession numbers NP_002181 and AAT22064. Refers to the mature form (ie, residues 24-155) and naturally occurring variants and polymorphs thereof. ii) As used herein, the term “rhIL-17A” refers to a recombinant derivative of native human IL-17A in which two additional amino acids (LE) are associated with the N-terminus of the mature form of native human IL-17A. Point to. This nomenclature is adopted for convenience when referring to various forms of IL-17A and may not match the usage in the literature. iii) As used herein, the term “FLAG-huIL-17A” refers to a variant of native human IL-17A with an associated N-terminal FLAG® peptide. In some experiments, FLAG-huIL-17A is biotinylated. iv) R & D System human IL-17A is herein residues 20-155 of human IL-17A protein accession number NP_002181 and AAT22064 with an additional N-terminal methionine. Table 1 summarizes the N-terminus of the IL-17A molecule variants referred to herein.

特段の記載が無い限り、アデノウィルスベクターを用いながら製造される本明細書に記載する実験において使用される何れのＩＬ−１７ＡもｒｈＩＬ−１７Ａである。「ＩＬ−１７Ａ」という用語は一般的にヒトＩＬ−１７Ａのネイティブ又は組み換えのもの、及びヒトＩＬ−１７Ａの非ヒト相同体を指す。特段の記載が無い限り、ＩＬ−１７Ａのモル濃度はＩＬ−１７Ａのホモ２量体の分子量を用いて計算される（例えばヒトＩＬ−１７Ａの場合は３０ｋＤａ）。

Unless otherwise noted, any IL-17A used in the experiments described herein that are produced using adenoviral vectors is rhIL-17A. The term “IL-17A” generally refers to the native or recombinant form of human IL-17A and the non-human homologue of human IL-17A. Unless otherwise noted, the molar concentration of IL-17A is calculated using the molecular weight of the IL-17A homodimer (eg, 30 kDa for human IL-17A).

As used herein, the term “antibody” refers to any form of antibody that exhibits the desired biological activity. That is, it is used in the broadest sense and includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies). As used herein, the term “IL-17A binding fragment” or “binding fragment” of an antibody (“parent antibody”) typically retains at least a portion of the binding specificity of the parent antibody. Antibody fragments or derivatives comprising at least part of the antigen binding or variable region (eg, one or more CDRs) of the parent antibody are included. Examples of antibody binding fragments include Fab, Fab ′, F (ab ′) ₂ , and Fv fragments; diabodies; linear antibodies; single chain antibody molecules such as sc-Fv; and multispecific antibodies formed from antibody fragments Including, but not limited to. Typically, a binding fragment or derivative retains at least 10% of its IL-17A binding activity when the activity is expressed on a molar basis. Preferably the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or higher IL-17A binding affinity of the parent antibody. It is also contemplated that IL-17A binding fragments can include conservative amino acid substitutions (referred to as “conservative variants” of the antibody) that do not substantially alter their biological activity. The term “binding compound” refers to both antibodies and binding fragments thereof.

The term “Fab fragment” includes one light chain and one heavy chain C _H 1 and variable regions. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

The “Fc” region contains two heavy chain fragments comprising the C _H 1 and C _H 2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain.

"Fab 'fragment" one light chain 1, and the VH domain and C _{H 1} domain, and further has contained the heavy chain one portion containing region between C _{H 1} and C _{H 2} domains, which Allows interchain disulfide bonds to be formed between two Fab ′ fragments and two heavy chains, thereby forming F (ab ′) ₂ molecules.

An “F (ab ′) ₂ fragment” contains two light chains and two heavy chains that contain a portion of the constant region between the C _H 1 and C _H 2 domains, so that the two heavy chains Interchain disulfide bonds are formed. That is, the F (ab ′) ₂ fragment consists of two Fab ′ fragments held together by a disulfide bond between the two heavy chains.

  “Fv region” includes variable regions derived from both heavy and light chains, but no constant regions.

The term “single chain Fv” or “scFv” refers to an antibody fragment comprising the V _H and V _L domains of an antibody, wherein these domains are present in a single polypeptide chain. In general, Fv polypeptides further comprise a polypeptide linker between the _VH and _VL domains that allows the scFv to form the desired structure for antigen binding. A discussion on scFv can be found in Plugthun (1994) THE PHARMALOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. Reference may also be made to International Patent Application Publication WO 88/01649 and US Pat. Nos. 4,946,778 and 5,260,203.

  A “domain antibody” is an immunologically functional immunoglobulin fragment that contains only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.

  A “bivalent antibody” contains two antigen binding sites. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies may be bispecific (see below).

  In the present specification, unless otherwise specified, “anti-IL-17A” antibody refers to human IL-17A or a variant thereof such as huIL-17A, rhIL-17A, FLAG-huIL-17A, and R & DIL-17A, Or an antibody raised against any antigenic fragment thereof.

  The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies that make up the population are naturally occurring, which may be present in small amounts. Except for possible mutations, they are identical. Monoclonal antibodies are highly specific and are directed against a single antigenic epitope. In contrast, traditional (polyclonal) antibody populations typically include many antibodies directed against (or specific to) different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be considered as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first reported by Kohler et al. (1975) Nature 256: 495, or may be produced by recombinant DNA methods (eg, US Pat. No. 4,816). 567). “Monoclonal antibodies” also refer to, for example, Clackson et al. (1991) Nature 352: 624-628 and Marks et al. Mol. Biol. 222: 581-597 may be used to isolate from a phage antibody library. Furthermore, Presta (2005) J. MoI. Allergy Clin. Immunol. 116: 731.

  The monoclonal antibodies described herein are particularly identical or homologous to corresponding sequences in antibodies in which a portion of the heavy and / or light chain is derived from a particular species or belongs to a particular antibody class or subclass. Chain residues include “chimeric” antibodies (immunoglobulins) that are derived from another species or are identical or homologous to the corresponding sequences in antibodies belonging to another antibody class or subclass, as well as fragments of such antibodies However, they must exhibit the desired biological activity (US Pat. No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855).

  As used herein, a “chimeric antibody” is an antibody having a variable domain derived from a first antibody and a constant domain derived from a second antibody, wherein the first and second antibodies are different species. Derived from. Typically, variable domains are derived from antibodies derived from laboratory animals such as rodents (“parent antibodies”), and constant domain sequences are derived from human antibodies, resulting in chimeric antibodies Will be less likely to exhibit an adverse immune response in human subjects than rodent antibodies.

  Monoclonal antibodies herein also include camelized single domain antibodies. See, for example, Muyldermans et al. (2001) Trends Biochem. Sci. 26: 230; Reichmann et al. (1999) J. MoI. Immunol. Reference may be made to Methods 231: 25; WO 94/04678; WO 94/25591; US Pat. No. 6,005,079, which are hereby incorporated by reference in their entirety. In one embodiment, the present invention provides a single domain antibody comprising two VH domains with modifications such that a single domain antibody is formed.

As used herein, the term “diabody” refers to a small antibody fragment having two antigen binding sites, which fragment is a heavy chain variable domain linked to a light chain variable domain (V _L ) within the same peptide chain. ( _VH ) ( _VH - _VL or _VL - _VH ). By using a linker that is too short to allow pairing between two domains on the same chain, the domain is forced to pair with the complementary domain of another chain and the two antigen binding sites Give rise to. Diabodies are described, for example, in EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a discussion of engineered antibody variants, see generally Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136.

  As used herein, the term “humanized antibody” refers to forms of antibodies that contain sequences from both human and non-human (eg, murine, rat) antibodies. Generally, a humanized antibody will comprise at least one, and typically substantially all of the two variable domains, in which case all or substantially all of the hypervariable loops are non-human immunoglobulin. And all or substantially all of the framework (FR) region will be of the human immunoglobulin sequence. The humanized antibody optionally comprises at least a portion (Fc) of a human immunoglobulin constant region.

  The antibodies of the present invention also include antibodies having an Fc region modified (or blocked) to provide altered effector function. Reference may be made, for example, to US Pat. No. 5,624,821; WO2003 / 086310; WO2005 / 120571; WO2006 / 0057702. Such modifications can be used to enhance or suppress various responses of the immune system and may have beneficial effects in diagnosis and therapy. Fc region modifications include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation, and addition of multiple Fc. Changes in Fc may also alter antibody half-life in therapeutic antibodies, allowing for a lower dosing frequency, thereby improving convenience and reducing material use. Presta (2005) J. MoI. Allergy Clin. Immunol. 116: 731, p. 734-35.

  The term “fully human antibody” refers to an antibody that comprises human immunoglobulin protein sequences only. Fully human antibodies may contain murine carbohydrate chains if produced in mice, mouse cells, or hybridomas derived from mouse cells. Similarly, “mouse antibody” refers to an antibody that comprises mouse immunoglobulin sequences only. Alternatively, fully human antibodies may contain rat carbohydrate chains if produced in rats, rat cells, or hybridomas derived from rat cells. Similarly, “rat antibody” refers to an antibody that comprises only rat immunoglobulin sequences.

  As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions are amino acid residues derived from “complementarity determining regions” or “CDRs” (ie, residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variable domain). And residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) in the heavy chain variable domain; Kabat et al. (1991) Sequences of Proteins of Immunological Health, 5th Ed. , National Institutes of Health, Bethesda, Md.) And / or residues derived from "hypervariable loops" (ie residues 26-32 (CDRL1), 50-52 (CDRL2) in the light chain variable domain) and 91-96 (CDRL3) and 26-32 (CDRH1), 53-55 (CDRH2) and 96-101 (CDRH3) in the heavy chain variable domain; Chothia and Lesk (1987) J. Mol. -917). As used herein, the term “framework” or “FR” residues refers to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

  “Binding agent” refers to a molecule, small molecule, macromolecule, antibody, fragment or analog thereof, or soluble receptor capable of binding to a target. A “binding agent” is also a complex of molecules, such as non-covalent complexes, ionized molecules, and, for example, phosphorylated, acylated, cross-linked, cyclized, or restricted, that can bind to a target. It may refer to a covalently or non-covalently modified molecule modified by cleavage. “Binding agent” may also refer to a molecule that can bind to a target in combination with a stabilizer, excipient, salt, buffer, solvent, or additive. “Binding” may be defined as the association of a binding agent with a target, where the association results in a reduction of the normal Brownian motion of the binding agent if the binding agent can be dissolved or suspended in solution.

  “Conservatively modified variants” or “conservative substitutions” are similar properties of amino acids in a protein (eg, charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and stiffness). Etc.), in which case changes can often be made without altering the biological activity of the protein. As is known to those skilled in the art, in general, substitution of a single amino acid in a non-essential region in a polypeptide does not substantially alter biological activity (eg Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed)). Furthermore, amino acid substitutions that are structurally or functionally similar are unlikely to disrupt biological activity. Various embodiments of the binding compounds of the present invention may be 0 (unchanged), 1, 2, 3, Polypeptide chains having sequences that include up to 4, 5, 6, 7, 8, 9, 10, 12, 15, 20 or more conservative amino acid substitutions are included. As used herein, the expression “up to X” conservative amino acid substitutions includes 0 substitutions and any number of substitutions up to X including X. Such exemplified substitutions are preferably made according to those shown in Table 2 below.

「本質的に〜よりなる」又はその変形「本質的に〜よりなる」又は「本質的に〜よりなっている」という用語は、明細書及び請求項を通じて使用する場合、何れかの言及された要素又は要素の群の包含、及び、特定される投薬の計画、方法又は組成物の基本的又は新規な特性を現実上変化させないような、言及した要素と同様又は異なる性質の別の要素の任意の包含を示す。非限定的な例として、本質的に言及したアミノ酸配列よりなる結合化合物は、結合化合物の特性に現実上影響しないアミノ酸１個以上を包含してよい。

The terms “consisting essentially of” or variations thereof “consisting essentially of” or “consisting essentially of” when used throughout the specification and claims refer to either The inclusion of an element or group of elements and any other element of similar or different nature to the element mentioned so as not to substantially change the basic or novel properties of the specified dosage regimen, method or composition Indicates the inclusion of As a non-limiting example, a binding compound consisting essentially of the amino acid sequence referred to may include one or more amino acids that do not actually affect the properties of the binding compound.

  An “effective amount” includes an amount sufficient to ameliorate or prevent a symptom or sign of a medical condition. Effective amount also means an amount sufficient to allow or facilitate diagnosis. Effective amounts for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the general condition of the patient, the route and dose of administration, and the severity of side effects (eg, US to Netti et al. Patent 5,888,530). An effective amount can be the maximum dose or dosing protocol that avoids significant side effects or toxic effects. The effect is at least 5%, usually at least 10%, more usually at least 20%, most usually at least 30 when the diagnostic scale or parameter improvement is defined as 100% of the diagnostic parameter exhibited by a normal subject. %, Preferably at least 40%, more preferably at least 50%, most preferably at least 60%, ideally at least 70%, more ideally at least 80%, and most ideally at least 90% (For example, Maynard et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interface Press, Boca Raton, FL; Dent (2001) Good Laboratory and Good Cl nical Practice, Urch Publ., London, see UK).

  “Exogenous” refers to substances produced outside an organism, cell, or human body, depending on the context. “Endogenous” refers to substances produced within a cell, organism, or human body, depending on the context.

  “Homology” refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences. If a position in both of the two compared sequences is occupied by monomeric subunits of the same base or amino acid, for example, if a position in each of the two DNA molecules is occupied by adenine, the molecule is Homologous at that position. The percent homology between two sequences is a function of 100 times the number of matching or homologous positions shared by the two sequences divided by the number of positions to be compared. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the 2 sequences are matched or homologous, the 2 sequences are 60% homologous. In general, comparisons are made while aligning the two sequences so that the maximum percent homology is obtained.

  “Immune condition” or “immune disorder” includes, for example, pathological inflammation, inflammatory disorders, and autoimmune disorders or diseases. "Immune condition" also refers to infections, refractory infections, and proliferative conditions such as cancer, tumors, and infections, tumors, and cancers that resist angiogenesis, such as eradication by the immune system. A “cancerous condition” includes, for example, cancer, cancer cells, tumors, angiogenesis, and precancerous conditions such as dysplasia.

  An “inflammatory disorder” is a disorder or pathological condition whose pathological features are in whole or in part, for example, due to a change in the number of immune system cells, a change in migration rate, or a change in activation. Means. Immune system cells are for example T cells, B cells, monocytes or macrophages, antigen presenting cells (APCs), dendritic cells, microglia, NK cells, NKT cells, neutrophils, eosinophils, mast cells, or It includes any other cell that is particularly relevant to immunology, such as cytokine-producing endothelial or epithelial cells.

  “Isolated binding compound” refers to the purified state of the binding compound, and in such a context the molecule is another biological molecule, such as a nucleic acid, protein, lipid, carbohydrate, or other substance, such as a cell. It means that substantially no crushed material and growth medium are contained. In general, the term “isolated” includes such substances unless they are present in an amount that substantially interferes with the experimental or therapeutic use of the binding compounds described herein. It is not intended to refer to the complete absence or absence of water, buffer, or salt.

  An “isolated nucleic acid molecule” is one in which an isolated polynucleotide is not associated with all or a portion of a naturally occurring polynucleotide, or is linked to a polynucleotide that is not naturally linked. Means genomic DNA or RNA, mRNA, cDNA, or of synthetic origin or some combination thereof. For the purposes of this disclosure, it should be understood that a specific nucleotide sequence “comprising a nucleic acid molecule” does not encompass an intact chromosome. An isolated nucleic acid molecule “comprising” a specific nucleic acid sequence includes, in addition to the specific sequence, coding sequences for up to 10, or even up to 20, or more other proteins or portions thereof. Alternatively, it may include operably linked regulatory sequences that control the expression of the coding region of the mentioned nucleic acid sequence and / or may include vector sequences.

  The expression “control sequence” refers to a DNA sequence necessary for the expression of an operably linked coding sequence of a particular host organism. For example, suitable control sequences for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

  A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, a DNA for a presequence or secretion leader is operably linked to DNA for that polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide, and the promoter or enhancer Is operably linked to the coding sequence if it affects the transcription of the sequence, or the ribosome binding site is operably linked to the coding sequence if it is positioned to promote translation. . In general, “operably linked” means that the linked DNA sequences are contiguous, and in the case of a secretory leader, are contiguous and in the reading phase. However, enhancers do not have to be contiguous. Ligation is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are routinely used.

  In this specification, the expressions “cell”, “cell line” and “cell culture” are used interchangeably and all such designations include progeny cells. That is, the words “transformants” and “transformed cells” include primary target cells and cultures derived therefrom without regard for the number of metastases. Furthermore, not all progeny cells need to be exactly identical in DNA content due to intentional or accidental mutation. Also included are mutant progeny cells that have the same function or biological activity as screened for in the original transformed cells. If a distinct notation is intended, it will be clear from the context.

  As used herein, “polymerase chain reaction” or “PCR” refers to a procedure or technique for amplifying a very small amount of a particular small piece of nucleic acid, RNA and / or DNA, for example as described in US Pat. No. 4,683,195. Point to. In general, in order to be able to design oligonucleotide primers, it is necessary that sequence information derived from the end of the region of interest or beyond is available, and such a primer faces the template to be amplified. Identical or similar in strand and sequence. The 5 'ends of the two primers should match the ends of the amplification products. PCR can be used to amplify specific RNA sequences, specific DNA sequences derived from total genomic DNA, and cDNA, bacteriophage, or plasmid sequences transcribed from total cellular RNA. See generally, Mullis et al. (1987) Cold Spring Harbor Symp. Quant. Biol. 51: 263; edited by Errich (1989) PCR TECHNOLOGY (Stockton Press, NY). As used herein, PCR is an example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample that includes the use of a known nucleic acid and a nucleic acid polymerase as primers to amplify or form a specific piece of nucleic acid, Considered not the only one.

  As used herein, the term “germline sequence” refers to an immunoglobulin DNA sequence that has not been rearranged. Any suitable source of unrearranged immunoglobulin may be used.

  “Inhibitor” and “antagonist” or “activator” and “agonist” are, for example, inhibitory or activating, respectively, with respect to activation of a ligand, receptor, cofactor, gene, cell, tissue, or organ, respectively. Refers to molecules that are sex. For example, a gene, receptor, ligand, or cellular modulator is a molecule that alters the activity of a gene, receptor, ligand, or cell, in which case the activity is altered in activation, suppression, or its regulatory properties. Can do. The modulator may function alone, or may use cofactors such as proteins, metal ions, or small molecules. Inhibitors are, for example, compounds that reduce, block, prevent, delay activation, inactivate, desensitize, or down regulate genes, proteins, ligands, receptors, or cells. Activators are, for example, compounds that increase, activate, promote, enhance activity, sensitize, or up regulate genes, proteins, ligands, receptors, or cells. Inhibitors may also be defined as compounds that reduce, block, or inactivate constitutive activity. An “agonist” is a compound that interacts with a target to induce or promote increased activation of the target. An “antagonist” is a compound that counteracts the action of an agonist. Antagonists prevent, reduce, suppress, or neutralize agonist activity. Antagonists may also prevent, suppress, or reduce the constitutive activity of a target, eg, a target receptor, even if no agonist is found.

  In order to determine the degree of inhibition, for example, a predetermined sample such as a protein, gene, cell, or organism or specimen is treated with a potential activator or inhibitor and a control sample in the absence of the inhibitor Compare with A control sample, i.e. a sample not treated with antagonist, is assigned a relative activity value of 100%. Inhibition has an activity value relative to the control of about 90% or less, typically 85% or less, more typically 80% or less, most typically 75% or less, generally 70% or less. More typically 65% or less, most commonly 60% or less, typically 55% or less, usually 50% or less, more usually 45% or less, most usually 40% or less, Preferably less than 35%, more preferably less than 30%, even more preferably less than 25%, and most preferably less than 25%. Activation has an activity value relative to the control of about 110%, typically at least 120%, more usually at least 140%, more usually at least 160%, frequently at least 180%, More often at least 2 times, most often at least 2.5 times, usually at least 5 times, more usually at least 10 times, preferably at least 20 times, more preferably at least 40 times, and most preferably It shall be achieved when it exceeds 40 times.

  The endpoint of activation or inhibition can be monitored as follows. For example, the response to activation, suppression, and treatment of cells, physiological fluids, tissues, organs, and animal or human subjects can be monitored by endpoints. Endpoints may include, for example, a predetermined amount or percentage of an indication of inflammation, tumorigenicity, or degranulation or secretion of cells, such as cytokines, toxic oxygen, or protease release. Endpoints include, for example, ion efflux or transport; cell migration; cell adhesion; cell proliferation; metastatic potential; cell differentiation; and phenotypic changes such as inflammation, apoptosis, transformation, cell cycle, or metastasis A predetermined amount of expression change may be included (eg, Knight (2000) Ann. Clin. Lab. Sci. 30: 145-158; Hood and Cheresh (2002) Nature Rev. Cancer 2: 91-100; Time et al. (2003) Curr. Drug Targets 4: 251-261; Robbins and Itzkowitz (2002) Med. Clin. North Am. 86: 1467-1495; Grady and Markowitz (2002) Anu. Rev. Genomics. um.Genet.3: 101-128; Bauer, etc. (2001) Glia36: 235-243; Stanimirovic and Satoh (2000) Brain Pathol.10: see 113-126).

  The endpoint of inhibition is generally 75% or less of the control, preferably 50% or less of the control, more preferably 25% or less of the control, and most preferably 10% or less of the control. In general, the endpoint of activation is at least 150% of the control, preferably at least 2 times the control, more preferably at least 4 times the control, and most preferably at least 10 times the control.

  “Ligand” refers to small molecules, peptides, polypeptides, and membrane-related or membrane-bound molecules, or complexes thereof, that can act as agonists or antagonists of the receptor. “Ligand” also encompasses substances that are not agonists or antagonists but can bind to receptors. Furthermore, “ligand” includes a membrane-bound ligand that has been changed to a soluble form of the membrane-bound ligand, eg, by chemical or recombinant methods. For convenience, when membrane bound on the first cell, the receptor is usually present on the second cell. The second cell may have the same or different entity as the first cell. The ligand or receptor may be completely intracellular, i.e. it may be present in the cytoplasm, nucleus, or some other intracellular compartment. The ligand or receptor may change its position, for example, from the intracellular compartment to the outer surface of the plasma membrane. The complex of ligand and receptor is referred to as a “ligand receptor complex”. When a ligand and receptor are involved in the signaling pathway, the ligand is in a location upstream of the signaling pathway and the receptor is in a location downstream.

  “Small molecule” is defined as a molecule having a molecular weight of less than 10 kDa, typically less than 2 kDa, preferably less than 1 kDa, and most preferably less than about 500 Da. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive resources, synthetic molecules, peptide mimetics, and antibody mimetics. As a therapeutic agent, small molecules are more permeable to cells, less prone to degradation, and less likely to elicit an immune response than larger molecules. Small molecules, such as antibodies and cytokine peptidomimetics, and small molecule toxins have been reported (eg, Casset et al. (2003) Biochem. Biophys. Res. Commun. 307: 198-205; Muyldermans (2001) J. Biotechnol. 74: 277-302; Li (2000) Nat. Biotechnol. 18: 1251-1256; Apostolopoulos et al. (2002) Curr. Med. Chem. 9: 411-420; Monfardini et al. (2002) Curr. Pharm. Domingues et al. (1999) Nat. Struct.Biol.6: 652-656; Sato and Sone (2003) Bioche. m.J. 371: 603-608; see US Patent 6,326,482 to Stewart et al.).

“Specific” or “selectively” binding when referring to a ligand / receptor, antibody / antigen, or other binding pair is the presence of the protein in a heterogeneous population of proteins and other biological materials. Refers to the binding reaction that determines That is, under certain conditions, a specific ligand binds to a specific receptor and does not bind in a significant amount to other proteins present in the sample. An antibody of the intended method or a binding compound derived from the antigen-binding site of an antibody is at least 2-fold higher than its affinity for any other antigen for that antigen, or a variant or mutein, preferably It binds with an affinity that is at least 10 times higher, more preferably at least 20 times higher, and most preferably at least 100 times higher. In a preferred embodiment, the antibody will have an affinity greater than about 10 ⁹ M ^{−1 as} determined, for example, by Scatchard analysis (Munsen et al. (1980) Analyt. Biochem. 107: 220-239).

  As used herein, the term “immunomodulating agent” refers to a natural or synthetic agent that suppresses or modulates the immune response. The immune response can be a humoral or cellular response. Immunomodulating agents include immunosuppressive or anti-inflammatory agents.

  An “immunosuppressive agent”, “immunosuppressive drug” or “immunosuppressive substance” is a therapeutic agent used herein in immunosuppressive therapy to suppress or prevent the activity of the immune system. Clinically, they prevent rejection of transplanted organs or tissues (eg bone marrow, heart, kidney, liver) and / or autoimmune diseases or diseases most likely of autoimmune origin (eg Used in the treatment of rheumatoid arthritis, myasthenia gravis, systemic lupus erythematosus, ulcerative colitis, multiple sclerosis). Immunosuppressive drugs include glucocorticoids; cytostatics; antibodies (biological response modifiers); drugs that act on immunophilins; other known chemotherapeutics used to treat proliferative disorders Classified as a drug. With respect to multiple sclerosis, in particular, the antibiotics of the present invention can be administered in combination with a new class of myelin-binding protein-like therapeutics known as copaxones.

  “Anti-inflammatory agent” or “anti-inflammatory drug” refers to both steroidal and non-steroidal therapeutics. Steroids, also known as corticosteroids, are drugs similar to cortisol, a hormone naturally produced by the adrenal glands. Steroids are used as the primary treatment for certain inflammatory conditions, such as systemic vasculitis (vascular inflammation); and myositis (muscle inflammation). Steroids are also inflammatory conditions such as rheumatoid arthritis (chronic inflammatory arthritis that occurs in joints on both sides of the body); systemic lupus erythematosus (pervasive disease caused by abnormal immune system function); Sjogren's syndrome (dry eye used to treat eye) and chronic disorders that induce dry mouth).

  Non-steroidal anti-inflammatory drugs, usually abbreviated as NSAIDs, are drugs with analgesic, antipyretic and anti-inflammatory effects that reduce pain, fever and inflammation. The term “non-steroid” is used to distinguish these drugs from steroids that have similar eicosanoid inhibitory anti-inflammatory effects (among a wide range of other effects). NSAIDs are: rheumatoid arthritis; osteoarthritis; inflammatory arthropathy (eg ankylosing spondylitis, psoriatic arthritis, Reiter syndrome); acute gout; dysmenorrhea; metastatic bone pain; Indications for migraine; postoperative pain; mild to moderate pain due to inflammation and tissue injury; fever; and symptomatic relief of renal colic. NSAIDs include salicylates, arylalkanoic acids, 2-arylpropionic acids (profens), N-arylanthranilic acids (phenamic acids), oxicams, coxibs, and sulfonanilides.

  Disease-modulating anti-rheumatic drugs (DMARDs) may often be administered in combination with NSAIDs. Commonly prescribed DMARDs include hydroxychloroquine / chloroquine, methotrexate, gold therapeutics, sulfasalazine, and azathioprine.

II. Antibodies Specific for Human IL-17A The present invention relates to engineered anti-IL-17A antibodies and various inflammatory, immune and proliferative disorders such as rheumatoid arthritis (RA), osteoarthritis, chronic Rheumatoid arthritis, inflammatory fibrosis (eg, scleroderma, pulmonary fibrosis, and cirrhosis), inflammatory bowel disorders (eg, Crohn's disease, ulcerative colitis, and inflammatory bowel disease), asthma (eg, allergic asthma) ), Its use to treat allergies, COPD, multiple sclerosis, psoriasis and cancer.

  Any suitable method for producing monoclonal antibodies may be used to produce the anti-IL-17A antibodies of the invention. For example, a recipient animal may be immunized with a linked or unlinked (eg, naturally occurring) form of IL-17A homodimer or a fragment thereof. Any suitable method of immunization can be used. Such methods can include the use of one or more of adjuvants, other immunostimulants, repeated booster immunizations, and immunization pathways.

  Any suitable form of IL-17A can be used as an immunogen (antigen) for the production of non-human antibodies specific for IL-17A, and the antibodies can be screened for biological activity. The inducing immunogen may be full-length mature human IL-17A, for example, a linked and naturally occurring homodimer, a single epitope or a peptide comprising multiple epitopes. The immunogen may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. Immunogens may be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen can be of genomic or non-genomic (eg, cDNA) origin. The immunogen-encoding DNA may be expressed using suitable gene vectors such as, but not limited to, adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, plasmids, and non-viral vectors such as cationic lipids.

  Any suitable method can be used to elicit an antibody response with the desired biological properties, for example, to suppress binding of IL-17A to its receptor. In some embodiments, antibodies are raised in mammalian hosts such as mice, rodents, primates, humans and the like. Techniques for producing monoclonal antibodies are described, for example, by Stites et al. (Eds.) BASIC AND CLINICAL IMMUNOLOGY (4th edition) Lange Medical Publications, Los Altos, CA and references cited therein; Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd edition) Academic Press, New York, NY; That is, the monoclonal antibody may be obtained by various techniques well known in the art. Typically, splenocytes from animals immunized with the desired antigen are immortalized, typically by fusing with myeloma cells. Kohler and Milstein (1976) Eur. J. et al. Immunol. 6: 511-519. Other methods of immortalization include transformation with Epstein-Barr virus, oncogenes, or retroviruses, or other methods known in the art. See, for example, Doyle et al. (Eds.) (1994 and periodic supplement) CELL AND TISSUE MULTIURE: LABORATORY PROCEDURES, John Wiley and Sons, New York, NY. Colonies arising from single immortalized cells are screened for the production of antibodies with the desired specificity and affinity for the antigen. The yield of monoclonal antibodies produced by such cells may be enhanced by various techniques such as injection into the peritoneal cavity of vertebrates.

  Another suitable technique involves selection of a library of antibodies on phage or similar vectors. See, for example, Huse et al., Science 246: 1275-1281 (1989); and Ward et al., Nature 341: 544-546 (1989). The antibodies of the invention may be used without modification, for example as parenteral rodent antibodies, or modified to facilitate their use as therapeutics in human subjects, such as chimeric or humanized antibodies. . In some embodiments, the antibody is covalently or non-covalently labeled with a substance that provides a detectable signal. A wide variety of labeling and conjugation techniques are known and have been extensively reported in both the academic and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent necent moieties, magnetic particles, and the like. The patents describing the use of such labels are U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149. And 4,366,241. In addition, recombinant immunoglobulins may be produced as described in Cabilly, US Pat. No. 4,816,567; and Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86: 10029-10033; or may be produced in transgenic mice, see Mendez et al. (1997) Nature Genetics 15: 146-156, as well as Abgenix and Medarex techniques.

Antibodies against a given fragment of IL-17A can be generated by immunizing an animal with a conjugate of the given fragment of IL-17A with a carrier protein. Monoclonal antibodies are produced from cells that secrete the desired antibody. These antibodies can be screened for binding to normal or defective IL-17A. These monoclonal antibodies will typically bind with a K _d of at least about 1 μM, more usually at least about 300, 30, 10, or 3 nM, preferably at least about 300, 100, 30, 10, 3 or 1 pM. Since the K _d value and affinity are inversely proportional, when referring to binding at a given K _d “below”, the affinity is at least as high as the presented value, ie at least as low as the stated value. Refers to binding at K _d . Binding affinity may be measured by ELISA (see Examples 5-6 below) or by Biacore® surface plasmon resonance spectral analysis, KinExA or ECL methods (see Example 7 below). Suitable non-human antibodies can also be found using the biological tests described in Examples 8-11 and 16-17 below.

  An example of a method for producing an anti-human IL-17A antibody of the present invention is described in Example 2.

III. Humanization of IL-17A Specific Antibodies Any suitable non-human antibody can be used as a source for the hypervariable region of the anti-IL-17A antibodies of the invention. Non-human antibody sources include, but are not limited to, rodents (eg, mice, rats), Rabbits (eg, rabbits), cows, and non-human primates. For the most part, humanized antibodies are derived from non-human species (donor antibodies) such as mice, rats, rabbits or non-human primates in which the recipient's hypervariable region residues have the desired specificity and affinity. A human immunoglobulin (recipient antibody) that is replaced with the hypervariable region residues of Furthermore, humanized antibodies may comprise residues that are not present in the recipient antibody, or in the donor antibody, such as modifications made to further define antibody performance of the desired biological activity. More detailed descriptions can be found in Jones et al. (1986) Nature 321: 522-525; Reichmann et al. (1988) Nature 332: 323-329; and Presta (1992) Curr, Op. Struct. Biol. 2: 593-596.

  Methods for recombinant manipulation and production of antibodies include, for example, Boss et al. (US Pat. No. 4,816,397), Cabilly et al. (US Pat. No. 4,816,567), Law et al. (European Patent Application Publication No. 438310) and Winter ( EP 239400).

  Amino acid sequence variants of the humanized anti-IL-17A antibody of the invention may be produced by introducing appropriate nucleotide changes into the DNA of the humanized anti-IL-17A antibody or by peptide synthesis. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, but the final construct must possess the desired properties. Amino acid changes may also alter the post-translational processing of humanized anti-IL-17A antibodies, such as when changing the number or position of glycosylation sites.

  One useful method for identifying residues or regions of a humanized anti-IL-17A antibody that are preferred positions for mutagenesis is referred to as “alanine scanning mutagenesis”. Cunningham and Wells (1989) Science 244: 1081-1085. By identifying a group of target residues (eg charged residues like Arg, Asp, His, Lys and Glu) and replacing them with neutral or negatively charged amino acids (most preferably alanine or polyalanine), IL Alter amino acid interaction with -17A. The residues that are functionally sensitive to alanine substitution are then clarified by introducing another amino acid substitution. In one embodiment, the effect of the mutation at a given target codon is examined by alanine scanning or random mutagenesis followed by activity and binding analysis of the resulting humanized anti-IL-17A antibody variant.

  Amino acid sequence insertions include amino- and / or carboxy terminal fusions ranging in length from a residue to a polypeptide containing 100 or more residues, and intrasequence insertions of single or multiple amino acid residues To do. Examples of terminal insertions include a humanized anti-IL-17A antibody having an N-terminal methionyl residue, or an antibody fused to an epitope tag. Other variants include fusions to the N- or C-terminus of enzymes or polypeptides that increase the serum half-life of the antibody. Another type of variant is an amino acid substitution variant. In these variants, at least one amino acid residue of the humanized anti-IL-17A antibody molecule is removed and a different residue is inserted at that position. The most advantageous sites for substitution mutagenesis include hypervariable loops, but FR modifications are also contemplated. Hypervariable region residues or FR residues involved in antigen binding are generally substituted in a relatively conservative manner.

  Other amino acid variants of the antibody alter the original glycosylation pattern of the antibody, for example, by eliminating one or more carbohydrate moieties and / or adding one or more glycosylation sites. Antibody glycosylation is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. The presence of either of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation involves the attachment of N-acetylgalactosamine, galactose or xylose to hydroxy amino acids, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxyserine may also be used.

  Glycosylation sites are added to the antibodies of the invention by insertion of one or more of the above-described tripeptide sequences (in the case of N-linked glycosylation sites) or the addition of one or more serine or threonine residues (in the case of O-linked glycosylation sites). Can be made. Nucleic acid molecules encoding amino acid sequence variants of humanized IL-17A specific antibodies are produced by a variety of methods known in the art. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants), or oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, or cassettes Includes by mutagenesis.

  Usually, an amino acid sequence variant of a humanized anti-IL-17A antibody has at least 50%, preferably at least 70% amino acid sequence identity with the original humanized antibody amino acid sequence, either heavy or light chain. 80%, 85%, 90%, and most preferably at least 95%. Identity or homology for this sequence aligns the sequences optimally (ie after achieving the maximum percent sequence identity by aligning the sequences and introducing gaps as needed) and any conservative substitutions. Is also defined herein as the percentage of amino acid residues in a candidate sequence that are identical to a humanized anti-IL-17A residue when not considered part of the sequence identity. None of N-terminal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence are considered to affect sequence identity or homology.

The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE. In one embodiment, the antibody is an IgG antibody. Any isotype of IgG, such as IgG ₁ , IgG ₂ , IgG ₃ , and IgG ₄ can be used. IgG isotype variants are also contemplated. A humanized antibody may comprise sequences from more than one class or isotype. Optimization of the constant domain sequences necessary to produce the desired biological activity is readily accomplished by scouring the antibody in the biological tests described below in the examples.

  Similarly, any class of light chain can be used in the compounds and methods described herein. In particular, kappa, lambda, or variants thereof are useful in the compounds and methods of the invention.

  Any suitable portion of a CDR sequence derived from a non-human antibody can be used to produce a humanized antibody of the invention. CDR sequences may be mutagenized by substitution, insertion or deletion, but such mutations are minimized because it is necessary to maintain the binding affinity and specificity of IL-17A. Typically, at least 75% of humanized antibody CDR residues will correspond to those of non-human CDR residues, more often 90%, and most preferably greater than 95%, and frequently 100% %. Any suitable portion of the FR sequence derived from a human antibody can be used. The FR sequence can be mutagenized by substitution, insertion or deletion of at least one residue so that the FR sequence is distinguishable from the human and non-human antibody sequences used. Such mutations are intended to be minimal. Typically, at least 75% of humanized antibody residues correspond to those of human FR residues, more often 90%, and most preferably greater than 95%.

  Chimeric antibodies or fragments thereof are also contemplated as long as they exhibit the desired biological activity (US Pat. No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855). As described above, a typical chimeric antibody contains a constant domain sequence derived from one type of antibody linked to the variable domain of an antigen-specific antibody obtained from a different species.

  The binding compounds of the invention may include bispecific antibodies. As used herein, the term “bispecific antibody” refers to an antibody, typically a monoclonal antibody, that has binding specificity for at least two different antigenic epitopes. In one embodiment, the epitopes are derived from the same antigen. In another embodiment, the epitope is derived from two different antigens. Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using the co-expression of two immunoglobulin heavy / light chain pairs. See, for example, Milstein et al. (1983) Nature 305: 537-39. Alternatively, bispecific antibodies can be produced using chemical linkages. See, for example, Brennan et al. (1985) Science 229: 81. Bispecific antibodies include bispecific antibody fragments. See, for example, Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-48; Gruber et al. Immunol. 152: 5368 (1994).

  An example of a method for humanizing the anti-human IL-17A antibody of the present invention is described in Example 3.

IV. Characterization of IL-17A Specific Antibodies As described in more detail in Examples 2 and 3, monoclonal antibodies that were immunoreactive with human IL-17A were generated using the methods described herein. 1A and 1B show the sequence alignments of the light and heavy chain variable regions of various anti-IL-17A antibodies of the present invention, respectively. CDR regions are shown and numbering was according to Kabat et al. (1991).

  A plasmid containing nucleic acid sequences encoding humanized 16C10 light chain and heavy chain was transferred to the American Type Culture Collection (ATCC-Manassas, Virginia, USA) under accession number PTA-7675 on July 28, 2006 in Budapest Treaty. Has been deposited in accordance with. Hybridomas expressing antibodies 30C10 and 23E12 were identified under the accession numbers PTA-7737 and PTA-7740 in the American Type Culture Collection (ATCC-Manassas, Virginia, USA) according to the Budapest Treaty on July 20, 2006, respectively. JL7-30C10. C3 and JL7-23E12. Deposited as B10.

The CDRs of the light chain and heavy chain of various humanized antibodies of the present invention are as shown in Tables 3 and 4, respectively. Furthermore, Table 4 shows the additional CDR regarding _{V H} of hu16C10 with variable position that can be used more than one amino acid.

一般的に、ヒト化抗体にかかわるＣＤＲは親ラット抗体のＣＤＲと同一であるが、ｈｕｍ１６Ｃ１０及びｈｕｍ４Ｃ３のＣＤＲＨ２及びＣＤＲＨ３は例外であり、これらは各々対応するラットＣＤＲからの単一のアミノ酸変化を有する。それらは独立したクローンとして得られたが、親ラット抗体１６Ｃ１０及び４Ｃ３はＶ_Ｈ領域の配列は同一であり、そしてＶ_Ｌ領域はフレームワーク置換のみ異なっている（１６Ｃ１０の１５位のイソロイシンは４Ｃ３ではバリンである）。その結果、ＣＤＲはこれらの２つの親ラット抗体に関して、従ってそれらのヒト化型に関しても同一である。

In general, the CDRs for a humanized antibody are identical to those of the parent rat antibody, with the exception of hum16C10 and hum4C3 CDRH2 and CDRH3, each having a single amino acid change from the corresponding rat CDR. . Although they were obtained as independent clones, the parent rat antibodies 16C10 and 4C3 are identical in V _H region sequence, and the _VL region differs only in framework substitution (isoleucine at position 15 of 16C10 is different in 4C3). Valin) As a result, the CDRs are the same for these two parent rat antibodies and thus for their humanized forms.

The sequences for the humanized _VL regions of antibodies 16C10 / 4C3 and 30C10 are shown in SEQ ID NOs: 5 and 22, respectively. The sequences for the humanized _VH regions of antibodies 16C10 / 4C3 and 30C10 are shown in SEQ ID NOs: 6 and 23, respectively. These humanized variable domains may be used to generate full length chimeric or humanized antibodies by adding appropriate constant domain sequences. Other embodiments include various other modifications at the CDR amino acid residues in the 16C10 heavy chain as shown in Table 4, for example. Referring to the residue numbering in FIG. 1B, the modifications listed in Table 4 (and in SEQ ID NOs: 17 and 20) are N54A, N54Q, N60A, N60Q, M96L, M96A, M96K, M96F, M100hF, M100hL. .

In one embodiment of the invention, the chimeric light and heavy chains of antibody 16C10 are human constant domains (each human kappa light chain at the C-terminus of the humanized V _L (SEQ ID NO: 5) and V _H regions (SEQ ID NO: 6). And a human IgG1 constant domain). The sequences of the chimeric 16C10 light chain and heavy chain are shown in SEQ ID NOs: 9 and 10. In other embodiments, the chimeric forms of antibodies 30C10 and 4C3 use the same constant domains from chimeric 16C10 with their corresponding humanized _VL and _VH regions (SEQ ID NOs: 22 and 23 for 30C10; It is prepared by fusing to SEQ ID NO: 5 and 6). The chimeric form of humanized 4C3 is of course identical to the chimeric form of humanized 16C10.

  In another embodiment, a full-length humanized antibody may contain the framework residues of the chimeric form of the antibody (ie, amino acid residues in the variable domain that are not part of the CDRs) as described in more detail in Example 3. Created by replacing cell lineage framework sequences. The resulting antibody retains only the CDR sequence derived from the rat antibody, and the constant domain and framework sequences are replaced by human derived sequences. The full length light and heavy chains for humanized antibody 16C10 including the signal sequence are shown in SEQ ID NOs: 2 and 4, respectively. In other embodiments, the humanized forms of antibodies 4C3 and 23E12 are in accordance with the methods described for 16C10, ie, replacing the appropriate human framework sequences with the chimeric sequences of these antibodies. (Above). See Example 3.

  In yet another embodiment, the full-length light and heavy chains of the humanized antibody of the invention promote secretion from the cell during antibody production by cloning with a signal peptide at their N-terminus. . In one embodiment, a 19 amino acid signal sequence is added to both the light and heavy chains of the humanized 16C10 antibody (residues -19 to -1 of SEQ ID NOs: 2 and 4). The humanized 16C10 full-length light and heavy chain DNA sequences to which a signal sequence has been added are shown in SEQ ID NOs: 1 and 3. Such DNA sequences can be cloned and expressed in any suitable expression vector for production of the humanized antibodies of the invention. In other embodiments, signal sequences may be added to the light and heavy chains of humanized antibodies 30C10 and 4C3 as described for antibody 16C10. In another embodiment, a signal sequence peptide different from the specific signal sequence shown in SEQ ID NOs: 1 to 4 is added depending on the intended method of antibody production. Such signal sequences may be obtained from academic literature such as Choo et al. (2005) “SPdb-a signal peptide database”, BMC Bioinformatics 6: 249.

In yet another embodiment, different constant domains may be added to the humanized _VL and _VH regions shown herein. For example, heavy chain constant domains other than IgG1 may be used if the particular intended use of the antibody (or puffer) of the present invention is to seek altered effector function. Although IgG1 antibodies provide long half-life and effector functions, such as complement activation and antibody-dependent cellular cytotoxicity, such activity may not be desirable for all uses of the antibody. In such cases, for example, an IgG4 constant domain may be used.

V. Affinity and biological activity of humanized anti-IL-17A Antibodies having the properties identified herein as desirable in humanized anti-IL-17A antibodies measure binding affinity in vitro, in vivo, or This allows screening for inhibitory biological activity. To obtain an antibody that binds to the same epitope on human IL-17A that is bound by the antibody of interest (eg, that blocks the binding of a cytokine to its receptor) by routine cross-blocking Tests such as those described in ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Laboratories, Ed Harlow and David Lane (1988) can be performed. Alternatively, for example, Champe et al. (1995) J. MoI. Biol. Chem. 270: 1388-1394, epitope mapping can be performed to see if the antibody binds to the epitope of interest. Antibody affinity (eg, for human IL-17A) may be measured using standard methods, including those described in Example 7. Preferred humanized antibodies are those that bind to human IL-17A with a K _d value of about 100 nM (1 × 10 ⁻⁷ M) or less, preferably about 10 nM or less, more preferably about 1 nM. Further preferred are embodiments in which the antibody has a _Kd value of about 200 pM (2 × 10 ⁻¹⁰ M), 100 pM, 50 pM, 20 pM, 10 pM, 5 pM, or even 2 pM or less.

  Antibodies and fragments thereof useful in the compounds and methods of the invention include, but are not limited to, biologically active antibodies and fragments. As used herein, the term “biologically active” refers to an antibody or antibody fragment that can bind to a desired antigenic epitope and exert a biological effect, either directly or indirectly. Typically, these effects are due to the inability of IL-17A to bind to its receptor. As used herein, the term “specific” refers to the selective binding of an antibody to a target antigen epitope. Antibodies can be tested for binding specificity by comparing binding to IL-17A under predetermined set conditions to binding to an irrelevant antigen or antigen mixture. An antibody is considered specific if it binds to IL-17A with an affinity that is at least 10-fold, preferably 50-fold higher than its affinity for an irrelevant antigen or antigen mixture. An antibody that “specifically binds” to a protein containing IL-17A (or a fragment thereof) does not bind to a protein that does not contain an IL-17A-derived sequence, ie, “specificity” is used herein as It relates to IL-17A specificity and is not any other sequence that may be present in the protein of interest. For example, as used herein, an antibody that “specifically binds” to FLAG-hIL-17A, a fusion protein comprising IL-17A and FLAG® peptide, is FLAG® peptide alone, or It does not bind when it is fused to a protein other than IL-17A.

  The data presented in the examples below (eg, Example 7) show that the humanized antibody 16C10 (including N54Q and M96A substitutions relative to the parent rat heavy chain CDR) has a high affinity for binding to human IL-17A. And has a Kd in the range of 1-10 pM as measured by KinExA analysis. In vitro activity tests such as Ba / F3hIL-17Rc-GCSFR cell proliferation test (Example 11), normal human skin fibroblast (NHDF) test (Example 9), and human rheumatoid arthritis (RA) synovial cell test ( According to Example 8), for hu16C10, the observed IC50 value was typically less than 50% of the concentration of hIL-17A present during the test (100 pM, 1000 pM and 1000 pM in 3 tests, respectively) Thus, it is confirmed that the antibody is a high affinity antibody. The divalent properties of the antibody used in the experiment and the possibility of dimerization of IL-17A make it possible to achieve 50% inhibition of the predetermined concentration of IL-17A with less than 0.5 molar equivalents of antibody. According to in vivo activity tests, eg administration to mice with collagen-induced arthritis (Example 16) and BAL neutrophil recruitment test (Example 17), several anti-IL-17A antibodies of the invention in animals Activity has been confirmed. In vitro and in vivo activity studies have also confirmed that humanized antibody 16C10 is a neutralizing antibody, which was not known from binding experiments alone.

  The reason that several of the antibodies of the present invention are advantageous in their ability to bind to cynomolgus IL-17A as well as human IL-17A is that such potential therapeutic antibodies may have separate cynomolgus monkey specific antibodies for toxicological testing. This is because it can be used directly for such tests in cynomolgus monkeys without the need to develop. Several high affinities of the antibodies of the present invention may also reduce the required dose in human (and other) subjects, which is advantageous in that it also reduces the likelihood of certain adverse reactions. Furthermore, high affinity may reduce the volume to be administered to a subject and reduce treatment costs.

  The serum half-life of hu16C10 has been measured in mice and cynomolgus monkeys. In cynomolgus monkeys, half-life after intravenous (iv) administration was evaluated in dose range studies using dosages of 0.4, 4.0 and 40 mg / kg. The serum concentration of the drug was measured periodically for 42 days. The half-life for subcutaneous (sc) administration in cynomolgus monkeys was measured at a dosage of 4 mg / kg and was also followed for 42 days. The half-life in cynomolgus monkeys was 10-19 days for iv and 28 days for sc as measured by the end-of-life slope of the drug concentration vs time profile. Certain anomalous data points at higher doses were excluded from the analysis. Similar experiments in mice showed that the hu16C10 antibody has a half-life of 13-25 days for iv and 12-22 days for sc.

  Example 19 is used to measure the epitope bound by the exemplified anti-IL-17A antibody (16C10) of the present invention, ie, residues in the region of L74-Y85 of human IL-17A (SEQ ID NO: 40). Explains the method. Since the biological test data presented herein reveals that antibody 16C10 is a high affinity neutralizing antibody, other antibodies that bind to the same epitope are also neutralizing antibodies, and possibly the same Are expected to have a high binding affinity. The epitopes measured herein are obtained by functional measurement rather than structure determination, and the epitope reported herein differs in detail from epitopes determined by structural methods There is. The epitopes reported herein include at least some, but not all, of the amino acid residues that are important for antibody 16C10 binding. Epitopes bound by the antibodies of the invention may also be examined by other methods, such as cross-blocking experiments (see Example 12) or by structural methods such as X-ray crystal structure measurements. Additional antibodies that bind to the same epitope as antibody 16C10 may be obtained, for example, by screening antibodies raised against hIL-17A or by immunizing animals with peptides containing the epitope sequence.

V. Antibody Production For recombinant production of antibodies, the nucleic acid encoding it is isolated and inserted into a replicable vector for subsequent cloning (DNA amplification) or for expression. The DNA encoding the monoclonal antibody is easily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes capable of specifically binding to the genes encoding the antibody heavy and light chains). by). Many vectors can be used. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. In one embodiment, both the light and heavy chains of the humanized anti-IL-17A antibody of the invention are expressed from the same vector, such as a plasmid or an adenoviral vector.

  The antibody of the present invention may be produced by any method known in the art. In one embodiment, the antibody is a mammalian or insect cell in culture, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) 293 cells, mouse myeloma NSO cells, baby hamster kidney (BHK) cells. , Expressed in Spodoptera frugiperda ovary (Sf9) cells. In one embodiment, antibodies secreted from CHO cells are recovered and purified by standard chromatographic methods such as protein A, cation exchange, anion exchange, hydrophobic interaction, and hydroxyapatite chromatography. The resulting antibody is concentrated and stored in 20 mM sodium acetate pH 5.5.

  In another embodiment, the antibody of the present invention is produced in yeast according to the method described in WO2005 / 040395. Briefly, a vector encoding an individual light or heavy chain of an antibody of interest can be obtained from yeast Pichia pastoris, which is auxotrophic, optionally supplemented by different yeast haploid cells, such as yeast haploid cells. Introduce into different mating types. The transformed haploid yeast cells are then mated or fused to obtain diploid yeast cells capable of producing both heavy and light chains. Here, diploid strains can secrete fully assembled biologically active antibodies. The relative expression levels of the two strands can be expressed, for example, using different copy number vectors, using different strength transcription promoters, or from inducible promoters that drive transcription of genes encoding one or both strands. Can be optimized by inducing

  In one embodiment, one expressing a plurality of light chains by introducing each heavy and light chain of a plurality of different anti-IL-17A antibodies ("original" antibodies) into yeast haploid cells. A library of mating type haploid yeast strains and a library of different mating type haploid yeast strains expressing multiple heavy chains are generated. By crossing (or fusing as spheroplasts) these libraries of haploid strains, a series of diploid yeast cells expressing a combinatorial library of antibodies consisting of various possible permutations of light and heavy chains are obtained. can get. The combinatorial library of antibodies can then be screened to determine whether any of the antibodies have properties superior to those of the original antibody (eg, higher affinity for IL-17A). For example, reference can be made to WO2005 / 040395.

In another embodiment, the antibody of the invention is a human domain antibody in which portions of the antibody variable domain are linked in a polypeptide having a molecular weight of about 13 kDa. See for example US Patent Publication 2004/0110941. Such single domain low molecular weight drugs offer many advantages in terms of ease of synthesis, stability, and route of administration.
VI. Pharmaceutical Composition and Administration To produce a pharmaceutical or sterile composition of the anti-huIL-17A antibody of the present invention, the antibody is mixed with a pharmaceutically acceptable carrier or excipient. See, for example, Remington's Pharmaceutical Sciences and the United States Pharmacopeia: National Formula, Mack Publishing Company, Easton, PA (1984).

  The preparation of therapeutic and diagnostic agents is made by mixing physiologically acceptable carriers, excipients or stabilizers, for example in the form of lyophilized powders, slurries, aqueous solutions or suspensions. (E.g. Hardman et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington, 2000). , New York, NY; Avis et al. (Ed.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al. (Eds.) (1990) Pharmaceutical Dosage Forms: Tablet, Marcel Dekker, NY; Lieberman et al. (Eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY). In one embodiment, an anti-IL-17A antibody of the invention is diluted to an appropriate concentration in a sodium acetate solution of pH 5-6 and NaCl or sucrose is added for osmotic adjustment. Additional materials such as polysorbate 20 or polysorbate 80 may be added to enhance stability.

Toxicity and therapeutic effects of antibody compositions administered alone or in combination with other agents are, for example, LD ₅₀ (dose critical to 50% of the population) and ED ₅₀ (50% of the population are therapeutically effective). Can be measured by standard pharmaceutical procedures in cell cultures or laboratory animals to measure a certain dose). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD ₅₀ / ED ₅₀ ). Antibodies that exhibit high therapeutic indices are preferred. The data obtained from these cell culture studies and animal studies can be used to set a range of doses for use in humans. Such dosage of the compound lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dose may vary within this range depending on the dosage form used and the route of administration.

  The mode of administration is not particularly important. Suitable routes of administration include oral, rectal, transmucosal, or enteral; parenteral delivery such as intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intraventricular, intravenous, intraperitoneal, nasal Includes intra- or intra-ocular injection. Administration can be carried out in various conventional ways, for example by oral ingestion, inhalation, absorption, topical application, or by skin, transdermal, subcutaneous, intraperitoneal, parenteral, intraarterial or intravenous injection. Intravenous administration to the patient is preferred.

  Alternatively, the antibody can be administered locally rather than systemically, for example, in an arthritic joint characterized by immunopathology or pathogen-induced lesions, frequently cumulative injections or sustained release formulations Injecting the antibody directly. Furthermore, administration of the antibody in a targeted drug delivery system, eg, in a liposome coated with a tissue specific antibody targeting an arthritic joint characterized by immunopathology or a pathogen-induced lesion. Good. Liposomes are targeted to and affected by the affected tissue.

  The mode of administration depends on several factors, such as the turnover rate of the therapeutic antibody in serum or tissue, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target cells in the biological matrix. Depends on. Preferably, the mode of administration delivers sufficient therapeutic antibody to result in an improvement of the target disease state while at the same time minimizing undesirable side effects. Thus, the amount of biopharmaceutical delivered depends in part on the particular therapeutic antibody and the severity of the condition to be treated. Guidance in selecting appropriate doses of therapeutic antibodies is available (eg, Wawrzynczak (1996) Antibiotic Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) Clina (ed.) , Marcel Dekker, New York, NY; Bach (eds.) (1993) Monoclonal Antibodies and Peptide Therapeutics in Autoimmune Diseases, N60, N3. Mi Grom et al. (1999) New Engl. J. Med. 341: 1966-1973; Slamon et al. (2001) New Engl. J. Med. 344: 783-3792; Beniaminovitz et al. (2000) New Engl. J. Med. 613-619; Ghosh et al. (2003) New Engl. J. Med. 348: 24-32; Lipsky et al. (2000) New Engl. J. Med. 343: 1594-1602).

  The determination of an appropriate dose is made by a physician using, for example, parameters or factors known or believed to affect treatment in the art. Generally, the dose begins with an amount somewhat less than the optimum dose and is gradually increased by small increments until the desired or optimum effect is achieved, relative to any undesirable side effects. Important diagnostic measures include, for example, a measure of the symptoms of inflammation, or the level of inflammatory cytokines produced. Preferably, the biopharmaceutical to be used is derived from the same species as the animal targeted for treatment, thereby minimizing an inflammatory, autoimmune or proliferative response to the reagent . For human subjects, for example, chimeric, humanized, and fully human antibodies are preferred.

  Antibodies, antibody fragments, and cytokines can be provided by continuous infusion or by doses administered, for example, daily, 1-7 times weekly, weekly, biweekly, monthly, bimonthly, and the like. The dose may be provided by intravenous, subcutaneous, topical, oral, nasal, rectal, intramuscular, intracerebral, intraspinal, or inhalation. The total dose per week is generally at least 0.05 μg / kg body weight, more typically at least 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.25 mg / kg kg, 1.0 mg / kg, 2.0 mg / kg, 5.0 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg, or higher (see, eg, Yang et al. (2003) New Engl. Med. 349: 427-434; Herold et al. (2002) New Engl. J. Med. 346: 169-1698; Liu et al. Cancer Immunol.Immunother.52: 133-14 Reference). The dose may also achieve a predetermined target concentration of anti-IL-17A antibody in the subject's serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg / ml or higher. May be provided. In another embodiment, the humanized anti-IL-17A antibody of the invention is 10, 20, 50, 80, 100, 200, 500, 1000, or 2500 mg / subject weekly, biweekly, or “every 4 weeks”. It is administered subcutaneously or intravenously.

  As used herein, “suppressing” or “treating” or “treatment” includes delaying the onset of symptoms associated with a disorder and / or reducing the severity of symptoms of such disorder. The term further encompasses ameliorating existing uncontrollable or undesirable symptoms, preventing additional symptoms, and improving or preventing the underlying cause of such symptoms. That is, the term refers to having beneficial results for a vertebrate subject having a disorder, disease or condition or having the potential to develop such a disorder, disease or condition.

  As used herein, the terms “therapeutically effective amount”, “therapeutically effective dose” and “effective amount” are used to refer to a disease or condition when administered to a cell, tissue, or subject alone or in combination with another therapeutic agent. Refers to the amount of an IL-17A binding compound of the invention that is effective to prevent or ameliorate the progression of one or more symptoms or such disease or condition. A therapeutically effective dose further binds enough to provide symptom improvement, eg, treatment, cure, prevention or amelioration of the relevant medical condition, or an increase in the rate of treatment, cure, prevention or amelioration of such condition Refers to the amount of compound. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied in combination, a therapeutically effective dose refers to the combined amount of active ingredients that provides a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of the therapeutic agent will improve the diagnostic measure or parameter by at least 10%, usually at least 20%, preferably at least 30%, more preferably at least 40%; and most preferably at least 50%. .

  Methods for co-administration of a second therapeutic agent such as a cytokine, another therapeutic antibody, a steroid, a chemotherapeutic agent, or an antibiotic are well known in the art. For example, Hardman et al. (Eds.) (2001) Goodman and Gilman's The Pharmacological Basis of The Atropetic Therapeutics, 10th edition, McGraw-Hill, New York, NY; Poole and Peter (NY); , Lippincott, Williams & Wilkins, Phila, PA; Caber and Longo (ed.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila. it can. The pharmaceutical composition of the present invention may also contain an immunosuppressant or immunomodulator. Any suitable immunosuppressive agent can be used, such as anti-inflammatory agents, corticosteroids, cyclosporine, tacrolimus (ie FK-506), sirolimus, interferon, soluble cytokine receptors (eg sTNRF and sIL-1R), Examples include, but are not limited to, agents that neutralize cytokine activity (eg, infliximab, etanercept), mycophenolate mofetil, 15-deoxyspergualin, thalidomide, glatiramer, azathioprine, leflunomide, cyclophosphamide, methotrexate, and the like. The pharmaceutical composition can also be used with other therapeutic means such as phototherapy and radiation.

  The IL-17A binding compounds of the invention may also be combined with one or more antagonists (eg, antibodies) of other cytokines such as, but not limited to, IL-23, IL-1β, IL-6, and TGF-β. It can also be used. See, for example, Veldhoen (2006) Immunity 24: 179-189; Dong (2006) Nat. Rev. Immunol. 6 (4): 329-333. In various embodiments, the IL-17A binding compounds of the invention are administered before, simultaneously with, or after administration of other antagonists. In one embodiment, the IL-17A binding compounds of the invention are used alone or in combination with IL-23 antagonists in the acute early treatment of adverse immune responses (eg, MS, Crohn's disease). In the latter case, the IL-17A binding compound may be gradually reduced and the suppression of adverse responses is maintained by continuing treatment with the IL-23 antagonist alone. Alternatively, antagonists to IL-1β, IL-6 and / or TGF-β may be administered simultaneously with, before or after the IL-17A binding compound of the invention. Cua and Kastelein (2006) Nat. Immunol. 7: 557-559; Tato and O'Shea (2006) Nature 441: 166-168; Iwakura and Ishigame (2006) J. MoI. Clin. Invest. 116: 1218-1222.

  Typical veterinary, laboratory, or research subjects include monkeys, dogs, cats, rats, mice, rabbits, guinea pigs, horses, and humans.

VII. Uses The present invention provides methods for using engineered anti-IL-17A antibodies for the treatment and diagnosis of inflammatory disorders and conditions, as well as autoimmune and proliferative disorders. Inflammatory bowel disease (IBD), multiple sclerosis (MS), chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), psoriasis, systemic scleroderma, autograft rejection, autoimmune myocarditis And methods for the diagnosis, prevention or treatment of peritoneal adhesions (see, eg, Chung et al. (2002) J. Exp. Med. 195: 1471-78).

Psoriasis The skin functions as an important boundary between the internal milieu and the environment while preventing contact with potentially harmful antigens. Upon infiltration of the antigen / pathogen, an inflammatory response is induced to eliminate the antigen. This response results in skin infiltration consisting primarily of T cells, polymorphonuclear cells, and macrophages (see, eg, Williams and Kupper (1996) Life Sci., 58: 1485-1507). Normally, this inflammatory response triggered by a pathogen is under tight control and will cease upon elimination of the pathogen.

  In certain cases, this inflammatory response occurs in the absence of external stimuli and in the absence of appropriate control, resulting in skin inflammation. The present invention provides a method for treating and diagnosing skin inflammation. Skin inflammation, the result of cell infiltration as described above and secreted cytokines from these cells, is the result of several inflammatory disorders such as scar pemphigoid, scleroderma, purulent scab, toxic epidermis Includes necrosis, pressure ulcers, osteomyelitis, graft versus host disease (GvHD), pyoderma gangrenosum, and Behcet's syndrome (eg, Williams and Griffiths (2002) Clin. Exp. Dermatol., 27: 585-590). reference). The most common form of skin inflammation is psoriasis.

  Psoriasis is characterized by enhanced T cell-mediated proliferation of keratinocytes associated with inflammatory infiltration. The disease has certain distinct and overlapping clinical phenotypes, such as chronic patchy lesions, skin rashes, and pustular lesions (eg, Gudjonsson et al. (2004) Clin. Exp. Immunol. 135: 1- 8). Approximately 10% of psoriasis patients develop arthritis. The disease is powerful but has a complex genetic predisposition and is 60% consistent with identical twins.

  A typical affected area of psoriasis is an erythema-like plaque with a clear border covered with thick silver scales. Inflammation and hyperproliferation of psoriatic tissue is associated with histological antigenicity and cytokine profiles that differ from normal skin. Among the cytokines associated with psoriasis are TNFα, IL-19, IL-18, IL-15, IL-12, IL-7, INFγ, IL-17A and IL-23 (see Gudjonsson et al., Above. Out). IL-17A has been detected in psoriatic skin.

  The anti-IL-17A antibodies of the present invention may be used alone or in combination with other agents in preventing, treating, diagnosing and predicting sudden exacerbation of psoriasis. The use of anti-IL-17A antibodies in predicting and treating the expansion of psoriasis is described in commonly assigned US Patent Application Publication No. 2005/0287593 and PCT Patent Publication No. WO 2005/108616, the disclosure of which is hereby incorporated by reference in its entirety. Incorporated in the description.

Rheumatoid arthritis (RA)
RA is a progressive systemic disease characterized by inflammation of the synovial joint that affects approximately 0.5% of the world population. Reference may be made to Emery (2006) BMJ 332: 152-155. Joint inflammation can result in deformation, pain, stiffness and edema, and ultimately irreversible deterioration of the joint. Affected joints include knee, elbow, neck and palm and leg joints. Traditional treatment uses NSAIDs to relieve symptoms, followed by administration of disease modifying anti-rheumatic drugs (DMARDs) such as gold, penicillamine, sulfarazine and methotrexate. Recent advances include treatment with TNF-α inhibitors such as monoclonal antibodies such as infliximab, adalmimab and golimumab, and receptor fusion proteins such as etanercept. Treatment with such TNF-α inhibitors dramatically reduces structural damage due to the disease.

  The anti-IL-17A antibodies of the present invention may be used in subjects in need of such treatment to treat RA. Example 16 describes an experiment using a collagen-induced arthritis (CIA) model of RA, the data for which are shown in FIGS. 3A-3D and Table 15. The results show that in animals treated with the anti-IL-17A antibodies of the present invention, the proportion of paw with high disease severity score was reduced compared to diluent and isotype controls.

  The anti-IL-17A antibodies of the invention may be combined with other treatments of RA, such as methotrexate, azathioprine, cyclophosphamide, steroids, mycophenolate mofetil, NSAIDs, or TNF-α inhibitors (antibodies or receptor fragments). Also good.

  In one embodiment, the anti-IL-17A antibodies of the invention are used to treat human subjects that have not previously had a satisfactory response to treatment with DMARD alone. In another embodiment, treatment with an anti-IL-17A antibody of the invention is initiated early in the course of the disease without requiring past failure of DMARD therapy. Such early intervention is appropriate if, for example, the safety of antibody therapy is firmly established.

  Clinical improvement is measured by measuring the ACR score, as described in more detail in Example 18. In various embodiments, ACR scores of 20, 50, and 70 are desirable endpoints, and these endpoints are any suitable time points in the course of treatment, such as weeks 5, 10, 15, 24, 40, 50 weeks. Or later.

Multiple sclerosis (MS)
MS is thought to be an autoimmune disease of the central nervous system (CNS) where loss of myelin from nerve fibers occurs, resulting in plaques or affected areas. The most common form is recurrent / relaxing MS, in which case symptomatic sudden exacerbations with a clear boundary occur, followed by a partial or complete relaxation period. Conventional treatment options are interferon-beta-1a and -1b, mitoxantrone, tetrapeptide glatiramer acetate, therapeutic alpha-4-integrin-specific antibody (natalizumab), or small molecule antagonism of alpha-4-integrin Agents (such as those disclosed in WO2003 / 084984).

  The anti-IL-17A antibodies of the present invention may be used in subjects in need of such treatment to treat MS. Anti-IL-17A antibodies may also be combined with other therapies for MS, such as interferon-beta, interferon-alpha, steroids, or alpha-4-integrin specific antibodies.

Inflammatory bowel disease (IBD)
IBD is the name of a group of disorders (eg, Crohn's disease and ulcerative colitis) where the intestine becomes inflamed resulting in abdominal cramps and pain, diarrhea, weight loss and intestinal bleeding. IBD affects more than 600,000 Americans. Conventional treatment options include sulfasalazine, corticosteroids (eg prednisone), immune system inhibitors such as azathioprine and mercaptopurine, or antibiotics (eg metronidazole) for Crohn's disease. Therapeutic monoclonal antibodies include etanercept, natalizumab and infliximab.

  The anti-IL-17A antibodies of the present invention may be used in subjects in need of such treatment to treat IBD. Yen et al. (2006) J. MoI. Clin. Invest. 116: 1310-1316; Fujimo et al. (2003) Gut 52: 65-70). The anti-IL-17A antibodies of the invention may be combined with other treatments for IBD, such as IL-10 (US Pat. Nos. 5,368,854, 7,052,686), steroids and sulfasalazine.

  In other embodiments, an antibody of the invention that does not block binding of IL-17A to its receptor (eg, non-neutralizing antibody 12E6) stabilizes IL-17A in a subject in need of long-term IL-17A activity. Use it therapeutically. Such subjects include patients suffering from infections or cancer.

  Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The invention is defined in terms of the full scope of the appended claims and the equivalents of such claims. The specific embodiments described herein, including the examples described below, are shown for purposes of illustration and are not intended to limit the scope of the invention in detail.

Example 1
General Methods Standard methods in molecular biology have been described (Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook 200 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA.). Standard methods are also described in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which also includes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3). ) And bioinformatics (Vol. 4).

  Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization have been described (Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, protein glycosylation have been described (eg Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New. Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17, Sigma-Aldrich, Co. 2001) Products for Life Science Research, St. Louis, MO., Pp. 45-89; Amersham Pharma cia Biotech (2001) BioDirectory, Piscataway, NJ, pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies have been described (Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using. (Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions can be used (see, eg, Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

  Monoclonal, polyclonal, and humanized antibodies can be produced (eg, Sheppard and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Universal. Press, New York, NY; -Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 139-243; 5: 6205; He et al. (1998) J. Immunol.160: 1029; Tang et al. (1999) J. Biol.Chem.274: 27371-27378; Baca et al. (1997) J.Biol. Chothia et al. (1989) Nature 342: 877-883; Foote and Winter (1992) J. Mol. Biol. 224: 487-499; US Pat. No. 6,329,511). An alternative to humanization is the use of human antibody libraries displayed on phage or human antibody libraries in transgenic mice (Vaughhan et al. (1996) Nature Biotechnol. 14: 309-314; Barbas (1995) Nature Medicine 1 : 837-839; Mendez et al. (1997) Nature Genetics 15: 146-156; Hoogenboom and Chames (2000) Immunol. Today 21: 371-377; Barbas et al. , Cold Spring Harbor NY; Kay, etc. (1996) Phage Display of Peptides and Proteins:. A Laboratory Manual, Academic Press, San Diego, CA; de Bruin, etc. (1999) Nature Biotechnol.17: 397-399).

  Single chain antibodies and diabodies have been described (eg, Malecki et al. (2002) Proc. Natl. Acad. Sci. USA 99: 213-218; Conrath et al. (2001) J. Biol. Chem. 276: 7346-7350; Desmyter et al. (2001) J. Biol. Chem. 276: 26285-26290; Hudson and Kortt (1999) J. Immunol. Methods 231: 177-189; and U.S. Pat. No. 4,946,778). . Bifunctional antibodies have been reported (e.g., Mack et al. (1995) Proc. Natl. Acad. Sci. USA 92: 7021-7025; Carter (2001) J. Immunol. Methods 248: 7-15; Volkel et al. (2001). ) Protein Engineering 14: 815-823; Segal et al. (2001) J. Immunol. Methods 248: 1-6; Brennan, et al (1985) Science 229: 81-83; Raso et al. (1997) J. Biol. 272: 27623; Morrison (1985) Science 229: 1202-1207; Traunecker et al. (1991) EMBO J. 10: 3655-3659; Pat.Nos.5,932,448,5,532,210, reference and 6,129,914).

  Bispecific antibodies have also been reported (eg, Azzoni et al. (1998) J. Immunol. 161: 3493; Kita et al. (1999) J. Immunol. 162: 6901; Merchant et al. (2000) J. Biol. Chem. 74. Pandey et al. (2000) J. Biol. Chem. 275: 38633; Zheng et al. (2001) J. Biol. Chem. 276: 12999; Propst et al. (2000) J. Immunol. 165: 2214; Ann. Rev. Immunol. 17: 875).

Purification of the antigen is not necessary for the production of antibodies. Animals can be immunized with cells carrying the antigen of interest. Spleen cells can then be isolated from the immunized animal and hybridomas can be produced by fusing the spleen cells with a myeloma cell line (eg, Mayaard et al. (1997) Immunity 7: 283-290; Wright et al. (2000). ) Immunity 13: 233-242; Preston et al., Supra; see Kaithana et al. (1999) J. Immunol. 163: 5157-5164). Antibodies usually at least about ^{10 -6} M, typically at least about ^{10 -7} M, more typically at least about ^{10 -8} M, preferably at least about ^{10 -9} M, and more preferably at least about 10, Will bind with a K _d of ⁻¹⁰ M, and most preferably at least about 10 ⁻¹¹ M (eg, Presta et al. (2001) Thromb. Haemost. 85: 379-389; Yang et al. (2001) Crit. Rev. Oncol). Hematol.38: 17-23; see Carnahan et al. (2003) Clin. Cancer Res. (Suppl.) 9: 3982s-3990s).

  The antibodies can be conjugated, for example, to small molecule drugs, enzymes, liposomes, polyethylene glycol (PEG). Antibodies are useful for therapeutic, diagnostic, kit or other purposes and include, for example, antibodies coupled to dyes, radioisotopes, enzymes, or metals such as colloidal gold (eg Le Dousal et al. ( 1991) J. Immunol. 146: 169-175; Gibellini et al. (1998) J. Immunol. 160: 3891-3898; Hsing and Bishop (1999) J. Immunol.162: 2804-2811; Everts et al. Immunol.168: 883-889).

  Methods for flow cytometry, such as fluorescence activated cell sorting (FACS), can be used (eg Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hobogen, NJ1G Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ For example, nucleic acids, eg, nucleic acid primers and probes, for use as diagnostic agents. Fluorescence suitable for modifying polypeptides and antibodies Drugs can be used (Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue, St.Louis, MO).

  Standard methods for histological examination of the immune system have been described (eg, Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt et al. (2000) Colts et al. of History, Lippincott, Williams, and Wilkins, Phila, PA; Louis et al. (2002) Basic History: Text and Atlas, McGraw-Hill, New York, NY).

  For example, software packages and databases for examining antigen fragments, leader sequences, protein folds, functional domains, glycosylation sites, and sequence alignments can be used (eg GenBank, Vector NTI® Suite (Informax, Inc, Bethesda , MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.); DeCypher® (Time Logic Corp., Crystal Bay, Nevada.); Menne et al. (2000) Bioinformatics 74: Bioinformatics 74; (2000) Bioinformatics Applications Note 16:74 -742; Wren et al. (2002) Comput. Methods Programs Biomed. 68: 177-181; von Heijne (1983) Eur. J. Biochem. 133: 17-21; von Heijne (1986) Nucleic Acids Res. 4690).

(Example 2)
Rat anti-human IL-17A monoclonal antibody A monoclonal antibody against human IL-17A was obtained as follows. Eight week old female Lewis rats (Harlan Sprague Dawley, Indianapolis, Indiana, USA) were given a series of injections of recombinant human IL-17A (rhIL-17A) that had been expressed from an adenoviral vector in HEK239 cells. Injections were made on days 0, 14, 32, 46 and 83.

  Day 0 injection was a subcutaneous injection of 50 μg rhIL-17A with intraperitoneal (ip) injection of Freund's complete adjuvant. The 25 μg rhIL-17Asc injections on days 14, 32 and 46 were accompanied by an ip injection of Freund's incomplete adjuvant. Day 83 injection was a combination of ip injection of 20 μg rhIL-17A in Freund's incomplete adjuvant and intravenous (iv) tail vein injection of rhIL-17A in saline.

Test blood collection was performed on day 53. On day 87, rat splenocyte fusion was split into 30 96-well plates using 1.6 × 10 ⁸ splenocytes and 1.8 × 10 ⁸ myeloma cells for a total of 1.13 × 10 ^5. The total number of cells per well.

  Primary screening of the resulting monoclonal antibodies (thousands) was performed by indirect rhIL-17A ELISA (see Example 5). In the secondary screening for the obtained antibody, neutralization of rhIL-17A-induced expression of murine IL-6 by ST2 (mouse stromal) cells and neutralization of rhIL-17A-induced proliferation of Ba / F3 hIL-17Rc: mGCSFR cells. went. Approximately 11 monoclonal antibodies were further examined after the first and second screening. Subsequent experiments were conducted to confirm that the candidate antibodies can bind to native huIL-17A, thereby confirming that they are useful for various therapeutic, diagnostic, and / or research purposes. Such screening may be performed by in vitro activity tests or using binding tests (eg indirect ELISA or sandwich ELISA) by in vivo activity tests, examples of which are as described herein.

(Example 3)
Humanization of rat anti-human IL-17A antibody Humanization of rat anti-human IL-17A monoclonal antibody 16C10 was performed essentially as described in WO2005 / 047324 and WO2005 / 047326, the disclosure of which is hereby incorporated by reference in its entirety. Embedded in the book. Briefly, by using human constant domains to replace the parent (rat) constant domain and selecting and using human germline sequences that are homologous to rat variable domain sequences, as described in more detail below. A human framework for rat CDRs was obtained.

Procedure for Selection of Human Germline Framework Sequences The following steps were used when selecting the appropriate germline framework sequences when humanizing the anti-human IL-17A antibody of the present invention.

1) The non-human _VL and _VH domains are cloned and sequenced and the amino acid sequence is determined.

Heavy chain 2) Compare non-human _VH sequences to a group of five human _VH germline amino acid sequences, one representative of subgroups IGHV1 and IGHV4, and three representatives of subgroup IGHV3. The V _H subgroup is M.M. -P. Lefranc (2001) “Nomenclature of the Human Immunoglobulin Heavy (IGH) Genes”, Experimental and Clinical Immunogenetics, 18: 100-116. Comparisons to the five germline sequences are performed as follows.

A) Assign non-human _VH sequence residue numbers according to Kabat et al. (1991).

B) Align non-human _VH sequences with each of the five human germline sequences. Since the V gene contains only _VH residues 1-94, only these residues are considered in the alignment.

C) Define complementarity determination (CDR) and framework (FR) regions within the sequence. CDRs and FRs are described in Kabat et al. (1991) (supra) and Chothia and Less (1987) "Canonical Structures for the Hypervariable Regions of Immunoglobulins", Journal of ul 9, 90, Journal of ul. Define. That is, the definitions are V _H CDR1 = 26 to 35, CDR2 = 50 to 65, CDR3 = 95 to 102.

  D) For each of the residue positions listed below (Table 1), a numerical score is assigned at each residue position where the non-human and human sequences are identical (IDENTICAL).

＊Ｃ．Ｃｈｏｔｈｉａ等（１９８９）”Ｃｏｎｆｏｒｍａｔｉｏｎｓ ｏｆ Ｉｍｍｕｎｏｇｌｏｂｕｌｉｎ Ｈｙｐｅｒｖａｒｉａｂｌｅ Ｒｅｇｉｏｎｓ”，Ｎａｔｕｒｅ３４２：８７７−８８３においてＣＤＲコンフォメーションに影響すると記載されている。

* C. Chothia et al. (1989) “Configurations of Immunoglobulin Hypervariable Regions”, Nature 342: 877-883, are described to affect CDR conformation.

  E) Add scores for all residue positions. The acceptor germline sequence is the one with the highest total score. If two or more germline sequences have the same score:

  1) If the non-human and human sequences are IDENTICAL among the following residue positions, 1 is added to the total score for each position: 1, 3, 5-23, 25, 36, 38, 40-43, 46 66, 68, 70, 72, 74, 75, 77, 79-90, 92 (maximum 49).

  2) The acceptor germline sequence has the highest total score. If two or more germline sequences still have the same score, either is accepted as an acceptor.

If a light chain III) V _L sequence is a member of the kappa subclass of V _L compares the non-human V _L sequence with four groups of human V _L kappa germline amino acid sequences. The four groups are Barbie and Lefranc (1998) “The Human Immunoglobulin Kappa Variable (IGKV) Genes and Joining (IGKJ) Segments”, Expr. And Imm 71. -P. Lefranc (2001) “Nomenclature of the Human Immunoglobulin Kappa (IGK) Genes”, Experimental and Clinical Immunogenetics, each of four established human VL subgroups of 18 representatives described in 18: 161-174. The four subgroups are also described in Kabat et al. (1991) pp. It corresponds to 4 subgroups described in 103-130. Comparisons to the four germline sequences are performed as follows.

A) Assign non-human _VL sequence residue numbers according to Kabat et al. (1991).

B) Align non-human _VL sequences with each of the four human germline sequences. Since the V gene only contains _VL residues 1-95, only these residues are considered in the alignment.

C) Define complementarity determination (CDR) and framework (FR) regions within the sequence. CDRs and FRs are described in Kabat et al. (1991) and Chothia and Less (1987) “Canonical Structures for the Hypervariable Regions of Immunoglobulins”, Journal of Molecules 19 as defined in Bio9. That is, the definitions are V _L CDR1 = 24 to 34, CDR2 = 50 to 56, CDR3 = 89 to 97.

  D) For each of the residue positions listed below (Table 2), a numerical score is assigned at each residue position where the non-human and human sequences are identical (IDENTICAL).

＊Ｃ．Ｃｈｏｔｈｉａ等（１９８９）”Ｃｏｎｆｏｒｍａｔｉｏｎｓ ｏｆ Ｉｍｍｕｎｏｇｌｏｂｕｌｉｎ Ｈｙｐｅｒｖａｒｉａｂｌｅ Ｒｅｇｉｏｎｓ”，Ｎａｔｕｒｅ３４２：８７７−８８３，１９８９においてＣＤＲコンフォメーションに影響すると記載されている。

* C. Chothia et al. (1989) “Configurations of Immunoglobulin Hypervariable Regions”, Nature 342: 877-883, 1989, are described as affecting the CDR conformation.

  E) Add scores for all residue positions. The acceptor germline sequence is the one with the highest total score. If two or more germline sequences have the same score:

  1) If the non-human and human sequences are IDENTICAL among the following residue positions, 1 is added to the total score for each position: 1, 3, 5-23, 35, 37, 39-42, 57, 59 ~ 61, 63, 65-70, 72-86, 88.

  2) The acceptor germline sequence has the highest total score. If two or more germline sequences still have the same score, any are accepted as acceptors. If the VL sequence is a member of the VL lambda subclass, the same procedure is performed using the human VL lambda germline amino acid sequence derived from the literature source cited above.

Humanization of anti-human IL-17A antibody For constant domain modification, the variable light and heavy chain domains of antibody 16C10 (rat anti-human IL-17AIgG1) were cloned and human kappa light chain (CL domain) and human IgG1 heavy Each was fused to a chain (CH1-hinge-CH2-CH3). This combination of rat variable domain and human constant domain comprises a chimeric form of antibody 16C10. The light chain and heavy chain sequences of this chimeric 16C10 are shown in SEQ ID NOs: 9 and 10, respectively.

Regarding modification of framework regions of variable domains. The amino acid sequence of the _VH domain of antibody 16C10 was compared to a group of five human _VH germline amino acid sequences, one representative of subgroups IGHV1 and IGHV4, and three representatives of subgroup IGHV3. The V _H subgroup is M.M. -P. Lefranc "Nomenclature of the Human Immunoglobulin Heavy (IGH) Genes", Experimental and Clinical Immunogenetics, 18: 100-116,2001. Antibody 16C10 scored highest against human heavy chain germline DP-71 in subgroup IV.

_{V L} sequences of 16C10 was of the kappa subclass. This sequence was compared to a group of four human _VL kappa germline amino acid sequences. The group of four is Barbie and M.M. -P. Lefranc (1998) "The Human Immunoglobulin Kappa Variable (IGKV) Genes and Joining (IGKJ) Segments", Experimental and Clinical Immunogen. -P. Lefranc “Nomenclature of the Human Immunoglobulin Kappa (IGK) Genes”, Experimental and Clinical Immunogenetics, 18: 161-174, 2001, each of four established human VL subgroups of representatives. The four subgroups are also Kabat et al. (1991) pp. It corresponds to 4 subgroups described in 103-130. Antibody 16C10 scored highest against human light chain germline Z-A19 in subgroup II.

  After the desired germline framework sequences were determined, plasmids encoding full length humanized variable heavy and light chains were generated. Substituting human framework residues for the framework residues of parent rat antibody 16C10 can be considered equivalent to grafting rat 16C10 CDRs onto the human framework sequences. The resulting antibody is referred to herein as “16C10 wt”, which indicates the presence of the same CDR as the parent rat 16C10, which is an optimized CDR (two single amino acid modifications It is distinguishable from the above. Both heavy and light chain variable domains were codon-optimized, synthesized, and inserted onto the constant domain to allow potentially optimal expression. Codon optimization that may improve the expression of the cloned antibody is purely arbitrary.

The humanized 16C10 wt antibody achieved greater chemical stability of the final humanized antibody by modifying at two CDR residues in addition to substitution of human constant domains and framework sequences. Two changes are indicated by bold amino acid residues in the “hu16C10” _VH sequence shown in FIG. 1B. Referring to the Kabat numbering shown in FIG. 1B, residue 54 of CDR2 is changed from N (asparagine) in the rat antibody to Q (glutamine) in the humanized antibody, thereby NG of residues 54-55. The possibility of formation of isoaspartate in the sequence can be reduced. The formation of isoaspartate may attenuate or completely eliminate the binding of the antibody to its target antigen. Presta (2005) J. MoI. Allergy Clin. Immunol. 116: 731-734. Furthermore, residue 96 of CDR3 is changed from M (methionine) in the rat antibody to A (alanine) in the humanized antibody, thereby reducing the antigen binding affinity and the molecular weight of the final antibody preparation. The potential for sulfur oxidation of methionine, which can contribute to heterogeneity, is reduced. See above. These single residue modifications can be represented as N54Q and M96A. The final humanized 16C10 antibody disclosed herein contains these two substitutions relative to the parent rat 16C10 CDR.

  In another embodiment of the invention, the chimeric (non-humanized) 16C10 antibody can be modified to incorporate the two single residue modifications described above for the humanized form, N54Q and M96A.

  The light and heavy chain amino acid sequences of humanized antibody 16C10 (hu16C10) are shown in FIGS. 2A and 2B, respectively, and in SEQ ID NOs: 2 and 4 (including the signal sequence). One embodiment of a nucleotide sequence encoding the light chain and heavy chain of hu16C10 is shown in SEQ ID NOs: 1 and 3. Another embodiment of a nucleotide sequence encoding the light and heavy chains of hu16C10 is shown in FIG. 5A (SEQ ID NO: 62) and FIG. 5B (SEQ ID NO: 63).

  For clarification purposes regarding nomenclature, the Kabat numbering system uses non-numerized amino acid residue designations (eg, VH) to account for variations in CDR and framework region lengths between various antibodies. It is important to recognize that residues 83a, 83b, 83c) are included. While this numbering system is advantageous in that it makes it easier to refer to the corresponding amino acid residues between different antibodies with different length CDRs, the sequence numbering (e.g. the sequence listing) by exact sequential numbers and In comparison, conflicting marks may occur regarding specific amino acid residues. In the present specification, amino acid residues are labeled by referring to “Kabat numbering”, for example, with reference to the corresponding sequence table unless otherwise specified.

  As another clarification regarding nomenclature, SEQ ID NOs: 2 and 4 (humanized 16C10) include the sequence of the N-terminal signal peptide (each first 19 residues), which are in the mature form of the antibody. Is removed. SEQ ID NOs: 1, 3, 62 and 63 include 57 nucleotides encoding a signal sequence. As used herein, the “mature” form of a protein refers to a protein that does not have a signal sequence.

  Humanized antibody 4C3 is produced by a method similar to that described above for antibody 16C10. The parent rat 4C3 antibody differs only in a single amino acid residue in the light chain framework region, and such framework regions are replaced with human germline framework sequences during humanization, so that the final The typical humanized 4C3 antibody sequence is identical to that of the humanized 16C10 antibody.

  Humanized antibody 30C10 is also made by a method similar to that described above for antibody 16C10. When determining the appropriate human framework sequence to be used, the parent rat 30C10 antibody is best against human heavy chain germline DP-46 in subgroup III and human light chain germline ZA19 in subgroup II. These framework sequences are substituted for rat framework sequences to have a value score. Humanized 30C10VL and VH sequences are shown in SEQ ID NOs: 22 and 23, respectively. In other embodiments, the potential for oxidation of methionine sulfur in the human 16C10 antibody is avoided by mutating one or more methionine residues of the rat 30C10 CDR. In particular, heavy chain residue 34 (in CDRH1) and / or light chain residue 30f (Kabat numbering, see FIG. 1A) is changed from methionine to another amino acid, such as alanine. Such antibodies are then screened to confirm that methionine substitution does not reduce IL-17A binding affinity to undesirable levels.

  Chimeric, humanized, and signal sequence-containing forms of antibody 12E6 are made using the methods described herein, analogously to producing such antibodies based on parent rat antibody 16C10. The light and heavy chain CDRs for the parent rat antibody 12E6 are shown in SEQ ID NOs: 34-36 and 37-39. Human constant domain and variable domain framework sequences are introduced as described above. In one embodiment, the potential for oxidation of methionine sulfur in the humanized 12E6 antibody is avoided by changing heavy chain residue 34 (in CDRH1) from methionine to another amino acid, such as alanine. The resulting antibodies are then screened to confirm that methionine substitution does not reduce IL-17A binding affinity to undesirable levels.

  Chimeric, humanized, and signal sequence-containing forms of antibody 23E12 are made using the methods described herein, analogously to producing such antibodies based on the parent rat antibody 16C10. The light and heavy chain variable domain sequences for the parent rat antibody 23E12 are shown in SEQ ID NOs: 44 and 46 (DNA) and 45 and 47 (amino acids). The CDRs for parent rat antibody 23E12 are shown in SEQ ID NOs: 48-50 (light chain) and 51-53 (heavy chain). Human constant domain and variable domain framework sequences are introduced into the parent rat antibody as described above.

Example 4
Fully human anti-IL-17A antibody A fully human anti-IL-17A monoclonal antibody is produced using a transgenic mouse carrying part of the human immune system rather than a mouse system. These transgenic mice are referred to herein as “HuMAbs” and represent the minilocus of the human immunoglobulin gene encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences. Contain endogenous mu and kappa chain loci with targeted mutations that inactivate (Lonberg et al. (1994) Nature 368 (6474): 856-859). Thus, mice show reduced expression of mouse IgM or kappa, and human heavy and light chain transgenes introduced in response to immunization undergo class switching and somatic mutation, resulting in high affinity Forms human IgGκ monoclonal antibodies (Lonberg et al. (1994), supra; discussion literature is Lonberg (1994) Handbook of Experimental Pharmacology 113: 49-101; Lonberg et al. (1995) International. Rev. Immunol. 13: 65-93. , And Harding et al. (1995) Ann. NY Acad. Sci 764: 536-546). Generation of HuMab mice is generally known in the art and is described, for example, Taylor et al. (1992) Nucleic Acids Research 20: 6287-6295; Chen et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993). Proc. Natl. Acad. Sci. USA 90: 3720-3724; Choi et al. (1993) Nature Genetics 4: 117-123; Chen et al. (1993) EMBO J. Biol. 12: 821-830; Tuaillon et al. (1994) J. MoI. Immunol. 152: 2912-2920; Lonberg (1994) Handbook of Experimental Pharmacology 113: 49-101; Taylor et al. (1994) International Immunology 6: 579-591; Lonberg et al. (1995) Intern. Rev. Immunol. 13: 65-93; and Fishwild et al. (1996) Nature Biotechnology 14: 845-851, the contents of which are hereby incorporated by reference in their entirety. US Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814 5, 874; 299; 5,770, 429 and 5,545, 807; and International Patent Application Publication Nos. WO 98/24884; WO 94/25585; WO 93/1227; WO 92/22645; and WO 92/03918. All of which are hereby incorporated by reference.

  To generate fully human monoclonal antibodies to IL-17A, HuMab mice are immunized with the antigenic IL-17A polypeptide described in Lonberg et al. (1994); Fishwild et al. (1996) and WO 98/24884. Preferably, mice are 6-16 weeks of age upon initial immunization. For example, a purified preparation of IL-17A can be used to immunize HuMab mice intraperitoneally. Mice can also be immunized with whole HEK293 cells that are stably transformed or transfected with the IL-17A gene. An “antigenic IL-17A polypeptide” may refer to an IL-17A polypeptide or any fragment thereof that elicits an anti-IL-17A immune response in HuMab mice.

  In general, HuMAb transgenic mice are first immunized intraperitoneally (IP) with antigen in complete Freund's adjuvant, followed by biweekly IP immunization (usually a total of 6) with antigen in incomplete Freund's adjuvant. To respond best. Mice are first immunized with cells expressing IL-17A (eg, stably transformed HEK293 cells), then with a soluble fragment of IL-17A, followed by alternating immunization with the two antigens. The immune response is monitored over the course of the immunization protocol while obtaining plasma samples by retroorbital bleeds. Plasma is screened for the presence of anti-IL-17A antibodies, for example by ELISA, and mice with sufficient antibody titers of immunoglobulin are used for fusion. Mice are boosted intravenously with antigen 3 days before sacrifice and splenectomy. 2-3 fusions for each antigen may be required. Several mice are immunized with each antigen. For example, a total of 12 HuMAb mice from the HCO7 and HCO12 lines are immunized.

Hybridoma cells that produce monoclonal fully human anti-IL-17A antibodies are hybridoma techniques originally developed by Kohler et al. (1975) (Nature 256: 495-497); Trioma techniques (Hering et al. (1988) Biomed. Biochim. Acta. 47: 211-216 and Hagiwara et al. (1993) Hum. Antibod. Hybridomas 4:15); human B cell hybridoma technique (Kozbor et al. (1983) Immunology Today 4:72 and Cote et al. Sci.USA.80: 2026-2030); and the EBV hybridoma approach (Cole et al. (1985), Monoclonal Antibodies an Cancer Therapy, Alan R.Liss, Inc., prepared by methods known generally in the art, such as pp77-96). Preferably, mouse splenocytes are isolated and fused with PEG to a mouse myeloma cell line based on standard protocols. The resulting hybridomas may then be screened for production of antigen specific antibodies. In one embodiment, a single cell suspension of splenic lymphocytes from an immunized mouse is 6 minutes of the quantity of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) using 50% PEG. Fusing to one of Cells are plated at approximately 2 × 10 ⁵ cells / mL in flat bottom microplates, then 20% fetal clonal serum, 18% “653” conditioned medium, 5% origen, 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0 Incubate for 2 weeks in selective medium containing 0.055 mM 2-mercaptoethanol, 50 units / mL penicillin, 50 mg / ml streptomycin, 50 mg / ml gentamicin and 1 × HAT (Sigma; HAT is added 24 hours after fusion). Two weeks later, the cells are cultured in a medium in which HAT is replaced with HT. Individual wells are then screened by ELISA for human anti-IL-17A monoclonal IgG antibody. When vigorous hybridoma has grown, the medium is usually observed after 10 to 14 days. Antibody-secreting hybridomas are re-plated, screened again, and if still positive for human IgG, anti-IL-17A monoclonal antibody is subcloned at least twice by limiting dilution. Stable subclones are then cultured in vitro to obtain small amounts of antibody in tissue culture for characterization.

In another embodiment, the anti-IL-17A antibody molecules of the invention are produced recombinantly (eg, in an E. coli / T7 expression system). In this embodiment, a nucleic acid encoding an antibody molecule of the present invention (V _H or V _L ) is inserted into a pET-based plasmid and It is expressed in the E. coli / T7 system. There are several methods for producing recombinant antibodies known in the art, including US Pat. No. 4,816,567, which is incorporated herein by reference. Antibody molecules may also be produced recombinantly in CHO or NSO cells.

(Example 5)
Indirect ELISA of anti-IL-17A monoclonal antibody
Binding of anti-human IL-17A monoclonal antibody to rhIL-17A is assessed using an indirect enzyme-linked immunosorbent assay (ELISA). Briefly, a fixed concentration of rhIL-17A is bound directly to the wells of the microplate. The monoclonal anti-IL-17A to be tested is then added to the rhIL-17A coated plate where the antibody is captured and quantified. A more detailed protocol is as follows.

A 96-well U-shaped MaxSorp plate is coated with 50 μl / well of rhIL-17A (0.5 μg / ml) in carbonate coating buffer (“test plate”). The carbonate coating buffer is 2.9 g / L NaHCO ₃ , 1.6 g / L Na ₂ CO ₃ , pH 9.4. Plates are coated and incubated overnight at 4 ° C. Monoclonal antibodies to be screened are serially diluted in duplicate over a row of V-shaped bottom plates to a final volume of 60 μl / well (“serial dilution plate”). The test plate is washed 3 times with PBS-Tween in a plate washer (Skanwasher, Molecular Devices, Sunnyvale, California, USA) and dry blotted. PBS-Tween is obtained by adding 0.5 ml / L Tween 20 to 1 × PBS. Transfer 50 μL from each well of the serial dilution plate to the test plate and incubate for 1 hour at 25 ° C. The secondary antibody is diluted 1/2000 in diluent (PBS-BSA-Tween, ie PBS-Tween containing 1 g / L BSA). The secondary antibody against the rat monoclonal antibody is goat anti-rat IgG (H + L) (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pennsylvania, USA). The secondary turn for chimeric and humanized monoclonal antibodies is F (ab ′) ₂ goat anti-human IgG Fcγ-HRP (Jackson ImmunoResearch Laboratories, Inc.). Wash the test plate as before. Diluted secondary antibody (100 μl / well) is added to the appropriate wells in the test plate and the plate is incubated for 45 minutes at 25 ° C. Wash the test plate as before. ABTS (100 μl / well) (Kirkegaard & Perry Laboratories, Gaithersburg, Maryland, USA) was added and the plates were incubated for 5-10 minutes at 25 ° C., after which the absorbance was read from the plate reader (Versamax, Molecular Devices, Alandis, USA). Read at 405 nm above with shaking for 5 seconds before reading.

  Indirect ELISA results for various forms of anti-16C10 of the present invention are shown in Table 5. Binding is reported as EC50 (concentration of antibody required to obtain half the maximum signal). The results show that binding is detected in all forms of 16C10. Although such an indirect ELISA test is useful for rapid determination of the presence or absence of anti-IL-17A antibodies, the EC50 values obtained are test dependent and are typically absolute for any given antibody. It is not used to assess overall binding affinity.

（実施例６）
抗ＩＬ−１７Ａモノクローナル抗体のＥＬＩＳＡ
ｒｈＩＬ−１７Ａへの抗ヒトＩＬ−１７Ａモノクローナル抗体の結合を以下の通りＥＬＩＳＡを用いて試験する。手短にいえば、捕捉抗体をマイクロプレートのウェルに結合させ、その後固定濃度のｒｈＩＬ−１７Ａを添加する。次に試験すべきモノクローナル抗ＩＬ−１７Ａをプレート上の結合ｒｈＩＬ−１７Ａに対して滴定することにより最大の結合の半分を達成するために必要な抗体の濃度を求める。より詳細なプロトコルは以下の通りである。

(Example 6)
Anti-IL-17A monoclonal antibody ELISA
Binding of anti-human IL-17A monoclonal antibody to rhIL-17A is tested using an ELISA as follows. Briefly, capture antibody is bound to the wells of the microplate, followed by addition of a fixed concentration of rhIL-17A. The concentration of antibody required to achieve half maximal binding is then determined by titrating the monoclonal anti-IL-17A to be tested against the bound rhIL-17A on the plate. A more detailed protocol is as follows.

A 96-well microplate is coated with 100 μl / well of capture antibody (rat anti-hIL-17A12E6, 0.5 μg / ml) in carbonate coating buffer pH 9.5 (“test plate”). Plates are coated and incubated at 4 ° C. for 24-48 hours. The test plates are washed 3 times in a plate washer (Skan Washer, Molecular Devices, Sunnyvale, California, USA) and dry blotted. The plate is then placed on an orbital shaker at 25 ° C. for 1 hour with ELISA test buffer (20 mM Tris-HCl, 0.15 M NaCl, pH 7.4, 0.5% BSA, 0.05% Tween-20, 2 mM EDTA). Block with 200 μl / well. Plates were washed and adenovirus derived rhIL-17A or E. coli. 100 μl / well of E. coli derived human IL-17 (IL-17A) (R & D Systems, Minneapolis, Minnesota, USA) (0.1 μg / ml) was added to the ELISA test buffer and on an orbital shaker at 25 ° C. Incubate for 2 hours. The plate is washed and the monoclonal antibody to be screened is serially diluted over a 7-well column in the range of 1000 ng / ml to 0.0813 ng / ml using a 4-fold serial dilution. The plate is incubated for 1.5 hours at 25 ° C. on an orbital shaker. Plates were washed and 100 μl / well of secondary antibody (F (ab ′) ₂ goat anti-human kappa light chain-HRP, 1: 20,000 dilution, BioSource, Carlsbad, California, USA), except test blank wells Add. The plate is washed twice with the plate rotation between cycles (ie 2 cycles with 3 washes per cycle). TMB substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Maryland, USA) is added at 100 μl / well and incubated on an orbital shaker for 3-5 minutes. Stop solution is added (100 μl / well) and the absorbance of the plate is read at 450-570 nm on a plate reader (Versamax, Molecular Devices, Sunnyvale, California, USA).

  Table 6 shows the results of ELISA of various forms of antibody 16C10 of the present invention. Binding is reported as EC50 (concentration of antibody required to obtain half the maximum signal). The results show that binding is detected in all forms of 16C10. The values shown with the error range represent the average of multiple measurements with standard deviation.

（実施例７）
抗ヒトＩＬ−１７Ａ抗体の結合試験
電気ケミルミネセンス（ＥＣＬ）試験を用いたラット及びキメラ抗ヒトＩＬ−１７Ａ抗体の結合の測定
ＩＧＥＮ，Ｉｎｃ．（Ｇａｉｔｈｅｒｓｂｕｒｇ，Ｍａｒｙｌａｎｄ，ＵＳＡ）により開発されたオリゲン電気ケミルミネセンス技術を用いてＦＬＡＧ−ｈｕＩＬ−１７Ａに対するラット抗ヒトＩＬ−１７Ａ抗体（及び１つのキメラ抗体）の結合を計測した。Ｅｌｅｃｓｙｓ（登録商標）イムノアッセイシステム、Ｒｏｃｈｅ Ｄｉａｇｎｏｓｔｉｃｓ（Ｉｎｄｉａｎａｐｏｌｉｓ，Ｉｎｄｉａｎａ，ＵＳＡ）を参照できる。電気ケミルミネセンス技術は安定なルテニウムの金属キレート（Ｏｒｉ−ＴＡＧ）を使用し、これはトリプロピルアミン（ＴＰＡ）の存在下で、電圧適用時に電気ケミルミネセンスを発生する。直径ミクロン単位の常磁性ビーズが固相として機能し、急速な試験動態を促進する。ビーズ／複合体はフローセル中でチャネリングさせ、そして磁気の適用により電極において捕捉する。電圧を適用し、そして生じた電気ケミルミネセンスを計測する。

(Example 7)
Anti-human IL-17A antibody binding assay Measurement of rat and chimeric anti-human IL-17A antibody binding using an electrochemiluminescence (ECL) assay IGEN, Inc. Binding of rat anti-human IL-17A antibody (and one chimeric antibody) to FLAG-huIL-17A was measured using the origen electrochemiluminescence technique developed by (Gaithersburg, Maryland, USA). Reference may be made to the Elecsys® immunoassay system, Roche Diagnostics (Indianapolis, Indiana, USA). The electrochemiluminescence technique uses a stable ruthenium metal chelate (Ori-TAG), which generates electrochemiluminescence when voltage is applied in the presence of tripropylamine (TPA). Paramagnetic beads of micron diameter function as a solid phase, facilitating rapid test kinetics. The bead / complex is channeled in the flow cell and captured at the electrode by application of magnetism. A voltage is applied and the resulting electrochemiluminescence is measured.

  The ECL test is conducted as follows. A 3-fold serial dilution of anti-human IL-17A mAb in 50 μl of test buffer is made in a 96-well microplate to obtain a final concentration of 1-3 μg / ml in the first well. 50 μl of test buffer and 50 μl of 50 ng / ml biotinylated FLAG-huIL-17A are added to each well, followed by OriTag labeled goat anti-rat IgG (H + L) pAb (50 μl at 450 ng / ml) or anti-hIgG mAb (50 μl at 500 ng / ml) ) Is added. Finally, 50 μl of origen streptavidin-dyna beads are added to each well at 0.1 mg / mL. After 1 hour incubation at 25 ° C., the plate is processed on an Origen M Series M8 / 384 analyzer. Data is plotted using GraphPad prism software (GraphPad Software, San Diego, California, USA), and the calculated area under the curve is an approximate measure of binding.

  The results are shown in Table 7 (this partially includes duplicate measurements). Two rows showing rat 16C10 binding to FLAG-huIL-17A represent duplicate measurements. All rat anti-human IL-17A antibodies (1D10, 16C10, 30C10, 23E12) in the table bound to FLAG-huIL-17A as in the case of chimeric 16C10. All four antibodies also bound to cynomolgus IL-17A. Antibodies 16C10 and 30C10 did not bind to mouse IL-17A under the conditions of this test, whereas antibodies 1D10 and 23E12 did.

ＫｉｎＥｘＡ技術を用いたラット及びヒト化抗ヒトＩＬ−１７Ａ抗体に関する平衡解離定数（Ｋ_ｄ）の測定
抗ヒトＩＬ−１７Ａ抗体に関する平衡解離定数（Ｋ_ｄ）をＫｉｎＥｘＡ３０００機材（Ｓａｐｉｄｙｎｅ Ｉｎｓｔｒｕｍｅｎｔｓ Ｉｎｃ．，Ｂｏｉｓｅ，Ｉｄａｈｏ，ＵＳＡ）を用いて測定した。ＫｉｎＥｘＡは抗体、抗原及び抗体−抗原複合体の混合物中の未複合体化抗体の濃度の計測に基づいた速度論的排除試験法の原理を使用する。例えばＤａｒｌｉｎｇ ａｎｄ Ｂｒａｕｌｔ（２００４）Ａｓｓａｙ Ｄｒｕｇ Ｄｅｖ．Ｔｅｃｈｎｏｌ．２（６）：６４７−５７を参照できる。遊離抗体の濃度は極めて短時間固相固定化抗原に混合物を曝露することにより計測する。実際は、これは、フローセル中に捕獲された抗原コーティング粒子を通過して液相の抗原−抗体混合物を流動させることにより達成される。機材により発生したデータは専用ソフトウエアを用いて分析する。平衡定数は以下の仮定に基づいた数学的理論を用いて計算する。

KinExA Technology The equilibrium dissociation constant for the rat and humanized anti-human-IL-17A antibody was used _{(K d)} of measuring anti-human-IL-17A antibody regarding the equilibrium dissociation constant _{(K d)} KinExA3000 gear (Sapidyne Instruments Inc., Boise, Idaho, USA). KinExA uses the principle of a kinetic exclusion test based on measuring the concentration of unconjugated antibody in a mixture of antibody, antigen and antibody-antigen complex. See, for example, Darling and Brat (2004) Assay Drug Dev. Technol. 2 (6): 647-57. The concentration of free antibody is measured by exposing the mixture to a solid phase immobilized antigen for a very short time. In practice, this is accomplished by flowing the liquid phase antigen-antibody mixture through the antigen-coated particles captured in the flow cell. Data generated by the equipment is analyzed using dedicated software. The equilibrium constant is calculated using a mathematical theory based on the following assumptions.

  1. Binding follows the reversible binding equation for the following equilibrium:

k _on [Ab] [Ag] = k _off [AbAg], where K _d = k _off / k _on
2. Antibody (Ab) and antigen (Ag) bind 1: 1 and total antibody equals antigen-antibody complex (AbAg) + free antibody.

  3. The equipment signal is linearly related to the free antibody concentration.

  KinExA analysis was performed on several rat anti-human IL-17A antibodies, their humanized variants, and sequence variants of these humanized antibodies. IL-17A was induced from either human ("hu"), cynomolgus monkey ("cyno") or mouse ("mu"). IL-17A from the same species was used in both immobilization and solution phase for each KinExA measurement. Poly (methyl methacrylate) (PMAA) particles (98 micron) were coated with humans, mosquitoes, or mosquitoes according to the following: All experimental procedures were performed according to the KinExA3000 manual. All trials were performed in duplicate. Table 8 shows the conditions for KinExA.

抗原の２倍連続希釈物を製造し、一定濃度において抗体と混合した。混合物を２５℃で２時間インキュベートすることにより平衡化させた。

Two-fold serial dilutions of the antigen were made and mixed with the antibody at a constant concentration. The mixture was equilibrated by incubating at 25 ° C. for 2 hours.

  Table 9 shows the results of KinExA analysis. Molar concentrations for KinexA analysis are calculated based on molecular weights of 75 kDa for antibodies and 15 kDa for IL-17A to account for the presence of two binding sites on the antibody and the dimer nature of IL-17A. did. For some antibodies, duplicate experiments were performed using different batches of antibodies and / or antigens, in which case the mean values are shown in Table 9 with standard deviations. The binding constants for the humanized 16C10 wt antibody and the parental rat 16C10 antibody were similar at about 5-10 pM, indicating that humanization did not significantly reduce the high affinity of the parental rat 16C10 for human IL-17A. Humanized 16C10 incorporating various amino acid substitutions (N54Q, M96A, M100hF) including the final humanized 16C10 antibody (with N54Q and M96A substitutions compared to rat 16C10) was also tested and similar in the 1-10 pM range. It was found to have a high coupling constant. The Fab fragment of bound hu16C10 retained high affinity (16 pM) compared to the intact antibody. Other antibodies of the present invention (rat 1D10, rat 23E12, rat 30C10) also bound FLAG-huIL-17A and cynomolgus IL-17A with high affinity. Rat 1D10 bound to mice with an affinity of 10 pM similar to the affinity for human and cynomolgus monkey IL-17A, whereas rat 23E12 had a low affinity of 200-2000 for mouse IL-17A. (7000 pM). Antibodies 16C10 and 30C10 did not bind to mouse IL-17A (data not shown).

当該分野で知られた別の方法、例えばＢｉａｃｏｒｅ（登録商標）表面プラズモン共鳴スペクトル分析も本発明の抗体の親和性を計測するために使用してよい。Ｂｉａｃｏｒｅ（登録商標）分析を本発明の抗体の数種に対して実施したが、結合親和性が一般的に高値すぎるため正確に計測することができず、特に、解離速度はこの方法での計測には緩徐過ぎるものであった。しかしながらそのような分析は、より低い親和性の抗ＩＬ−１７Ａ抗体又はより速い解離速度定数を有する抗ＩＬ−１７Ａ抗体の分析においては有用である場合がある。

Other methods known in the art, such as Biacore® surface plasmon resonance spectral analysis, may also be used to measure the affinity of the antibodies of the invention. Biacore® analysis was performed on several of the antibodies of the present invention, but the binding affinity was generally too high to be measured accurately, and in particular, the dissociation rate was measured with this method. It was too slow. However, such analysis may be useful in the analysis of lower affinity anti-IL-17A antibodies or anti-IL-17A antibodies with faster dissociation rate constants.

(Example 8)
Synovial cell test for anti-IL-17A antibodies The ability of anti-IL-17A antibodies of the invention to block the biological activity of IL-17A (either rhIL-17A or native huIL-17A) is as follows: Measured by monitoring IL-17A-induced expression of IL-6 and IL-8 in primary cultures of synovial cells. Synovial cells are isolated by collagenase digestion of rheumatoid arthritis synovium obtained from knee replacement patients. Synoviocytes are grown in growth medium (DMEM, 10% BCS, 1 × Pen-Strep (50 IU / ml penicillin, 55 μg / ml streptomycin), 1 × beta mercaptoethanol (50 μM), 1 × glutamine (20 mM), 25 mM HEPES). Enrich by successive passages, freeze at passage 3 and store in liquid nitrogen. When ready for use in the test, the vial of cells is thawed, plated, and the cells are grown to near confluence. Cells are then passaged 1: 2 in larger flasks using trypsin / EDTA. Once sufficient cells have proliferated, the experiment begins by trypsinizing the cells, plating them into 96- or 48-well plates, and growing them to full confluence.

  IL-17A is diluted to 4 × with a final concentration of 120 ng / ml, ie 30 ng / ml (1 nM). IL-17A is human (rhIL-17A and native huIL-17A) or is derived from a non-human primate, in this case cynomolgus monkey. Add 100 and 300 μl aliquots of 4 × IL-17A stock solution to empty 96- and 48-well plates, respectively.

  The anti-IL-17A antibody to be tested is diluted in growth medium to the maximum concentration of 4 × to be tested in the experiment. A serial dilution of 4 × anti-IL-17A stock solution 1: 2 is made to encompass the suppression dynamic range of the test. 2 × concentrations of both IL-17A and anti-IL-17A antibodies by mixing 1: 1 each of serially diluted antibody samples (all at 4 × of their final concentration) with 4 × IL-17A solution in an empty plate. To form a mixture having These mixtures are allowed to equilibrate at 37 ° C. in a tissue culture incubator for more than 4 hours.

  The medium is removed from adherent confluent synovial cells and replaced with 100 μl (96-well plate) or 200 μl (48-well plate) growth medium. An equal volume of 2x ligand / 2x antibody solution is added to the synovial cells to give 1x IL-17A (final 30 ng / ml) and 1x antibody. Each well (data point) is tried in duplicate. Synovial cells are activated for 3 days (ie exposed to IL-17A / antibody mixture), at which point the supernatant is transferred to a 96-well plate, optionally frozen and stored at -80 ° C. until analysis. Thaw the microplate with the supernatant and dilute each solution 1:10 with growth medium. Supernatants are analyzed for IL-6 and IL-8 using Luminex bead pairs (Upstate, Charlottesville, Virginia, USA) according to the manufacturer's instructions.

  The results are shown in Tables 10 (IL-6) and 11 (IL-8). The numerical value shown with the error range shows the average of many measurements together with the standard deviation. Various types of antibodies 16C10, for example humanized forms of 16C10 with the original rat CDRs (“hu16C10 (wt)”), as well as several variants with one, two or three changes in the heavy chain CDRs (general To "hu16C10X ## Z", where X is the amino acid at residue ## in the heavy chain of hu16C10 (wt) and Z is a new amino acid). “NHPIL-17A” is non-human primate-derived IL-17A, in this case cynomolgus IL-17A. “Native huIL-17A” refers to mature huIL-17A that is produced when the precursor signal is produced using the native signal sequence and differs from rhIL-17A by the absence of the two N-terminal amino acids. ing. Concentration and IC50 values are displayed in ng / ml, but may be displayed in pM units. For example, 30 ng / ml rhIL-17A corresponds to 1000 pM (MW = 30 kDa) and 70 ng / ml anti-IL-17A antibody corresponds to about 470 pM (MW = 150 kDa).

（実施例９）
抗ＩＬ−１７Ａ抗体に関するＮＨＤＦ試験
ＩＬ−１７Ａの生物学的活性をブロックする本発明の抗ＩＬ−１７Ａ抗体の能力は、正常ヒト（成人）皮膚線維芽細胞（ＮＨＤＦ）初代細胞系統におけるＩＬ−６のｒｈＩＬ−１７Ａ誘導発現をモニタリングすることにより計測される。手短にいえば、種々の濃度の試験すべき抗ＩＬ−１７Ａ抗体をｒｈＩＬ−１７Ａとともにインキュベートし、そして得られた混合物を次にＮＨＤＦ細胞培養物に添加する。その後ＩＬ−１７Ａ活性を阻害する対象抗体の能力の尺度としてＩＬ−６生産を測定する。より詳細なプロトコルを以下に示す。

Example 9
NHDF Test for Anti-IL-17A Antibodies The ability of the anti-IL-17A antibodies of the present invention to block the biological activity of IL-17A is IL-6 in normal human (adult) dermal fibroblast (NHDF) primary cell line. It is measured by monitoring rhIL-17A-induced expression. Briefly, various concentrations of the anti-IL-17A antibody to be tested are incubated with rhIL-17A and the resulting mixture is then added to the NHDF cell culture. IL-6 production is then measured as a measure of the ability of the subject antibody to inhibit IL-17A activity. A more detailed protocol is shown below.

A 2-fold serial dilution of the anti-IL-17A antibody of interest is prepared starting from a 40 μg / ml stock solution (in duplicate). A stock solution of rhIL-17A is prepared at 120 ng / ml. 70 μl of rhIL-17A stock solution is mixed with 70 μl of anti-IL-17A antibody dilution in the wells of the microplate and incubated for 20 minutes at room temperature. Next, 100 μl of each of this mixture is added to the wells of a microplate that has been seeded with 1 × 10 ⁴ NHDF cells / well (100 μL) the previous night and incubated at 37 ° C. NHDF cells (passage 4) are obtained from Cambrex Bio Science (Baltimore, Maryland, USA). The final concentration of rhIL-17A is 30 ng / ml (1 nM), and the antibody ranges from 10 μg / ml to a one-half interval. After incubating the plate for 24 hours at 37 ° C., the supernatant is collected and 50 μL is taken for use in an IL-6 ELISA.

  An ELISA for detection of human IL-6 is performed as follows. Reagents are generally obtained from R & D Systems (Minneapolis, Minnesota, USA). The hIL-6 capture antibody (4 μg / ml solution 50 μl / well) is transferred to the wells of the microplate, which is sealed and incubated overnight at 4 ° C. The plate is washed 3 times and then blocked with 100 μl / well of blocking buffer for at least 1 hour. The plate is then washed again 3 times. Experimental samples (50 μl of culture supernatant) and controls (serial dilution of IL-6 protein) are added to the wells in 50 μl and incubated for 2 hours. The plate is washed 3 times and 50 μl / well of biotinylated anti-IL-6 detection antibody (300 ng / ml) is added. Plates are incubated for 2 hours at room temperature, washed 3 times, 100 μl / well of streptavidin HRP is added and incubated for 20 minutes. Plates are washed again, ABTS (BioSource, Carlsbad, California, USA) is added (100 μl / well) and incubated for 20 minutes. Stop solution is added (100 μl / well) and absorbance at 405 nm is measured.

  The IC50 for the anti-IL-17A antibody of interest is the concentration of antibody required to reduce the level of rhIL-17A-induced IL-6 production to 50% of the level observed in the absence of added anti-IL-17A antibody. is there.

  The results are shown in Table 12.

（実施例１０）
包皮線維芽細胞試験抗ＩＬ−１７Ａ抗体
ＩＬ−１７Ａの生物学的活性をブロックする本発明の抗ＩＬ−１７Ａ抗体の能力をＨＳ６８包皮線維芽細胞系統のｒｈＩＬ−１７Ａ誘導発現をモニタリングすることにより計測する。ｒｈＩＬ−１７Ａに応答したＩＬ−６の生産の低減を、本発明の抗ＩＬ−１７Ａ抗体によるブロッキング活性の尺度として使用する。

(Example 10)
Foreskin fibroblast test anti-IL-17A antibody The ability of an anti-IL-17A antibody of the present invention to block the biological activity of IL-17A was measured by monitoring rhIL-17A-induced expression of the HS68 foreskin fibroblast cell line. To do. Reduction of IL-6 production in response to rhIL-17A is used as a measure of blocking activity by the anti-IL-17A antibodies of the invention.

Analysis of IL-17RC (IL-17A receptor) expression in a panel of fibroblast cell lines has identified the human foreskin fibroblast line HS68 (ATCC CRL1635) as a potential IL-17A responsive cell line. This is a polyclonal goat anti-human IL-17R antibody (R & D Systems, Gaithersburg, Maryland, USA), followed by phycoerythrin (PE) -F (ab ') ₂ donkey anti-goat IgG (Jackson Immunoresearch, Inc., West Grove, Pennsylvanis, Pennsylvanies, Pennsylvania). , USA) and indirect immunofluorescence staining and analysis of PE immunofluorescence signals on a flow cytometer (FACScan, Becton-Dickinson, Franklin Lakes, New Jersey, USA). To further validate the model, IL-17A (both adenovirus-induced rhIL-17A and commercially available E. coli-derived IL-17A, R & D Systems) has an IL50 in HS68 cells with an EC50 of 5-10 ng / ml. Dose-responsive induction is induced, which is blocked by preincubation with commercially available polyclonal and monoclonal anti-IL-17A antibodies (R & D Systems).

The IL-17A suppression test is performed as follows. Confluent T-75 flasks (approximately 2 × 10 ⁶ cells) of HS68 cells were washed with Dulbecco's PBS without Ca ++ and Mg ++, then 2-5 minutes at 37 ° C. in an incubator at 5% CO ₂ . Incubated with 5 ml (Sigma-Aldrich, St. Louis, Missouri, USA). The cells were then harvested with 5 ml tissue culture (TC) medium and centrifuged at 1000 rpm for 5 minutes. The composition of the TC medium is Dulbecco's modified Eagle medium (added with glutamine), 10% heat-inactivated fetal calf serum (Hyclone), 10 mM Hepes, 1 mM sodium pyruvate, penicillin, and streptomycin. Cells were resuspended in 2 ml TC medium, diluted 1: 1 with trypan blue and counted. The cell concentration is adjusted to 1 × 10 ⁵ cells / ml in TC medium and aliquots of 0.1 ml / well are dispensed into wells of flat bottom plates containing 0.1 ml TC medium. Cells are grown overnight and the supernatant is aspirated and the cells are washed with 0.2 ml of fresh TC medium.

  A series of storages that can be used to serially dilute the anti-IL-17A to be tested in 2-fold or 3-fold steps to obtain a final antibody concentration of 1-0.001 μg / ml in the IL-17A inhibition test. Obtain a solution. Rat IgG control is used in each test, and spontaneous IL-6 production in HS68 cells is measured by using a medium only sample as a control. Aspirate TC medium from the wells of the plate containing HS68 cells. By preincubating aliquots (0.1 ml each) of various concentrations of anti-IL-17A antibody for 5 minutes at 37 ° C. with HS68 cells in wells, followed by addition of 0.1 ml of 20 ng / ml rhIL-17A A final concentration of 10 ng / ml rhIL-17A is obtained (IL-17A dimer approximately 330 pM). Cells are incubated at 37 ° C. for 24 hours and supernatants (50-100 μl) are collected and IL- using, for example, a human IL-6 ELISA kit available from Pharmingen (OptEIA-BD Biosciences, Franklin Lakes, New Jersey, USA). Test 6

  The results for several rat anti-human IL-17A antibodies of the present invention in the foreskin fibroblast IL-17A inhibition test are shown in Table 13.

（実施例１１）
Ｂａ／Ｆ３−ｈＩＬ−１７Ｒｃ−ｍＧＣＳＦＲ増殖試験
ＩＬ−１７Ａの生物学的活性をブロックする本発明の抗ＩＬ−１７Ａ抗体の能力を、ＩＬ−１７Ａ刺激に応答して増殖するように操作された細胞系統のｒｈＩＬ−１７Ａ誘導増殖をモニタリングすることにより計測する。特に、マウス顆粒球コロニー刺激因子受容体（ＧＣＳＦＲ）の膜貫通ドメイン及び細胞質領域に融合したヒトＩＬ−１７Ａ受容体（ｈＩＬ−１７ＲＣ）の細胞外ドメインを含む融合蛋白を発現するようにＢａ／Ｆ３細胞系統（ＩＬ−３依存性ネズミプロＢ細胞）を修飾した。得られた細胞系統は本明細書においてはＢａ／Ｆ３ ｈＩＬ−１７Ｒｃ−ｍＧＣＳＦＲと称する。細胞外ＩＬ−１７ＲＣドメインへのホモ２量体ＩＬ−１７Ａの結合はｈＩＬ−１７Ｒｃ−ｍＧＣＳＦＲ融合蛋白受容体の２量化をもたらし、これはＢａ／Ｆ３細胞の増殖を、それらのｍＧＣＳＦＲ細胞質ドメインを介してシグナルする。そのような細胞はＩＬ−１７Ａに応答して増殖し、抗ＩＬ−１７Ａ抗体のようなＩＬ−１７Ａ抑制剤に関する、好都合な試験法をもたらす。

(Example 11)
Ba / F3-hIL-17Rc-mGCSFR proliferation test Cells engineered to proliferate in response to IL-17A stimulation by the ability of the anti-IL-17A antibodies of the invention to block the biological activity of IL-17A Measure by monitoring rhIL-17A-induced proliferation of the strain. In particular, Ba / F3 so as to express a fusion protein comprising the transmembrane domain of mouse granulocyte colony stimulating factor receptor (GCSFR) and the extracellular domain of human IL-17A receptor (hIL-17RC) fused to the cytoplasmic region. A cell line (IL-3-dependent murine pro-B cells) was modified. The resulting cell line is referred to herein as Ba / F3 hIL-17Rc-mGCSFR. Binding of homodimeric IL-17A to the extracellular IL-17RC domain results in dimerization of the hIL-17Rc-mGCSFR fusion protein receptor, which leads to the proliferation of Ba / F3 cells via their mGCSFR cytoplasmic domain. Signal. Such cells proliferate in response to IL-17A, providing a convenient test for IL-17A inhibitors such as anti-IL-17A antibodies.

  Depending on the sensitivity of the Ba / F3-hIL-17Rc-mGCSFR proliferation test to IL-17A stimulation, the experiment should be performed at a relatively low concentration of rhIL-17A (eg 3 ng / ml, 100 pM) compared to other tests. However, a robust and easily measurable proliferative response is still maintained. This means that the concentration of anti-IL-17A antibody required to achieve a molar excess over rhIL-17A in the test is lower. Experiments performed at lower antibody concentrations allow discrimination between high affinity antibodies that are otherwise indistinguishable (ie, the experiment is a linear portion rather than a steady state portion of the antibody IL-17A binding curve). Can be implemented to approximate).

  Antibody and IL-17A were filtered through a 0.22 μm filter after dilution to working concentration but prior to addition to the experimental sample. Four sets of samples were made in duplicate across a row of 96-well flat bottom tissue culture plates. In this example, the growth medium is RPMI 1640 w / Glutamax (Invitrogen, Carlsbad, California, USA), 55 μM 2-mercaptoethanol, 10% mixed feed fed fetal bovine serum (Irvine Scientific, Santa Ana, California / US, USA / Ana, California / US) ml gentamicin, 2 μg / ml puromycin, and 10 ng / ml mIL-3. The bioassay medium is similar to the growth medium except that it does not contain puromycin and mIL-3. All serial dilutions in this example were performed in BioAssay Medium.

The following experimental samples (75 μl): 1) serial dilution of growth medium (containing 10 ng / ml of mIL-3), 2) serial dilution of rhIL-17A, 3) 3 ng / ml IL including “no antibody” control Serial dilutions of anti-IL-17A antibodies of the invention mixed with -17A (final concentration after addition of cells) and 4) "cells only" control without addition of IL-17A or mIL-3 antibody . Ba / F3hIL-17Rc-mGCSFR cells (7500 cells / well) were then added to bring the total volume to 100 μl / well and the plates were incubated for about 40 hours at 37 ° C./5% CO ₂ . AlamarBlue® indicator dye (11 μl / well) was added and the plates were incubated for 6-8 hours at 37 ° C./5% CO ₂ . The plate was then read for the difference in absorbance at 570 nm and 600 nm. IC50 values were determined using non-linear fit / sigmoidal dose response / variable slope.

  The results of the Ba / F3 hIL-17Rc-mGCSFR proliferation test are shown in Table 14.

（実施例１２）
抗ＩＬ−１７Ａ抗体の交差ブロッキング
本発明の異なる種類の抗ＩＬ−１７Ａ抗体は同じエピトープ、オーバーラップするエピトープ、又はオーバーラップしないエピトープ、例えば２つ以上の抗体が同時に１つのＩＬ−１７Ａ単量体に結合しうるに足ほど区別できるエピトープに結合する場合がある。受容体結合のために重要であるＩＬ−１７Ａの部分に結合する抗体はＩＬ−１７Ａの受容体媒介生物学的活性をブロックすることになる。そのような抗体は本明細書においては、「中和抗体」と称する。結合するが受容体結合をブロックしない抗体は非中和抗体と称する。

(Example 12)
Cross-blocking of anti-IL-17A antibodies Different types of anti-IL-17A antibodies of the present invention may be of the same epitope, overlapping epitopes, or non-overlapping epitopes, eg, two or more antibodies are simultaneously one IL-17A monomer In some cases, it binds to an epitope that can be separated as much as possible. Antibodies that bind to the portion of IL-17A that is important for receptor binding will block the receptor-mediated biological activity of IL-17A. Such antibodies are referred to herein as “neutralizing antibodies”. Antibodies that bind but do not block receptor binding are referred to as non-neutralizing antibodies.

  When conducting experiments on IL-17A and anti-IL-17A antibodies, it is useful to be able to examine the level of IL-17A (or anti-IL-17A) in a sample, such as by sandwich ELISA. See, for example, Example 6. In one format, an IL-17A ELISA involves coating the wells of a microplate with a capture antibody, adding an experimental sample suspected of containing IL-17A, and binding a detection antibody. The capture antibody and detection antibody must be able to bind to IL-17A simultaneously.

  A similar test may be used to measure the level of anti-IL-17A antibody, in which case a standard solution of IL-17A is bound to a well coated with a capture antibody, followed by an experiment believed to contain IL-17A. Sample is added and the secondary detection antibody is allowed to bind (eg, anti-human IgG antibody in the case of the IgG humanized antibody of the invention). As with the IL-17A sandwich ELISA, the capture antibody cannot interfere with the binding of the antibody to be tested.

  Preferred antibody pairs for use in the ELISA experiments outlined in this example can be determined by performing cross-blocking experiments. In a cross-blocking experiment, a first antibody is coated on the wells of a microplate. The biotinylated second antibody is then mixed with IL-17A and allowed to bind before adding the mixture to the coated wells and incubating. The biotinylated second antibody is added (ie, titrated) at various concentrations so that in at least some samples the antibody is present in a two-fold (or higher) molar excess than homodimeric IL-17A. You may ensure that. The plate is then washed and the presence or absence of biotinylated secondary antibody bound in the well is measured by standard methods.

  When the two antibodies cross-block, the plate in the presence of the second anti-IL-17A antibody has a signal (as compared to a control sample that does not contain the second anti-IL-17A antibody (or contains an isotype control) Decrease in IL-17A binding). Antibody pairs that do not cross-block can be used together in tests such as a sandwich ELISA. Use a pair of cross-blocking antibodies in an ELISA in certain formats (eg, when IL-17A binds to the capture antibody on the plate prior to the addition of the detection antibody) due to the dimer nature of IL-17A Although non-cross-blocking pairs of antibodies are generally preferred.

  Several anti-IL-17A antibodies of the invention (clone 4C3, 6C3, 8G9, 12E6, 16C10, 18H6, 23E12, 29H1, 30C10, 1D10, 21B12, 29G3) were used as a pair for cross-blocking. All pairs were cross-blocked except for 29G3 / 1D10 and 29G3 / 21B12, so these antibody pairs could be used in an ELISA. In addition to identifying pairs of anti-IL-17A antibodies that can be used in ELISA, these results indicate that the epitope bound by antibody 29G3 is functionally or physically distinct from the epitope bound by antibodies 1D10 and 21B12. It shows that it is possible. These data also reveal that the epitopes for 1D10 and 21B12 overlap but are not identical to the epitope for 16C10.

  Such pairs of anti-IL-17A antibodies that bind to functionally distinct epitopes are useful, for example, in enabling anti-IL-17A immunohistochemistry (IHC). For example, if a tissue sample shows the same pattern of IL-17A expression in IHC performed with two different anti-IL-17A antibodies that bind to a functionally distinct epitope, It is even more likely that IL-17A is being detected rather than other pseudo-reactive proteins.

  Such a non-cross-blocking antibody pair may also be used when designing an ELISA for detection of IL-17A in the presence of a therapeutic anti-IL-17A antibody in a sample from a patient undergoing anti-IL-17A antibody therapy, for example. In that case, the presence of an excess of therapeutic anti-IL-17A antibody will block detection by the anti-IL-17A ELISA unless the ELISA antibody is non-cross-blocking with the therapeutic antibody.

(Example 13)
Gene Therapy Using Anti-IL-17A Antibody The anti-IL-17A antibody of the present invention may also be administered to a subject by gene therapy. In the gene therapy research method, a target cell is transformed with a nucleic acid encoding the antibody of the present invention. The subject containing the nucleic acid then produces antibody molecules (intrabodies) endogenously. For example, Alvarez et al. Has introduced a single-chain anti-ErbB2 antibody into a subject using gene therapy research methods. Alvarez et al. (2000) Clinical Cancer Research 6: 3081-3087. The method disclosed by Alvarez et al. May be readily adapted for introduction of a nucleic acid encoding an anti-IL-17A antibody molecule of the invention into a subject. In one embodiment, the antibody molecule introduced by gene therapy is a fully human single chain antibody.

  The gene therapy approaches described herein have the potential advantage that if long-term gene expression is achieved, the treatment need only be performed once or at most a limited number of times. This is in contrast to antibody administration which must be repeated periodically to maintain an appropriate therapeutic level in the subject.

  The nucleic acid may be introduced into the subject cell by any means known in the art. In some embodiments, the nucleic acid is introduced as part of a viral vector. Examples of viruses that may be derived from vectors include lentiviruses, herpes viruses, adenoviruses, adeno-associated viruses (AAV), vaccinia viruses, baculoviruses, alphaviruses, influenza viruses, and other recombinant viruses with the desired cellular tropism Is included. Various companies manufacture commercially available viral vectors, see, for example, Avigen, Inc. (Alameda, CA; AAV vector); Cell Genesys (Foster City, CA; retrovirus, adenovirus, AAV and lentiviral vectors); Clontech (retrovirus and baculovirus vectors); Genovo, Inc. (Sharon Hill, PA; adenovirus and AVV vectors); Genvec (adenovirus vectors); IntroGene (Leiden, Netherlands; adenovirus vectors); Molecular Medicine (retrovirus, adenovirus, AAV, and herpes virus vectors); Norgen (adenovirus vectors) Oxford Biomedica (Oxford, United Kingdom; lentiviral vectors); and Transgene (Strasbourg, France; adenovirus, vaccinia, retrovirus, and lentiviral vectors).

  Methods for constructing and using viral vectors are known in the art (see, eg, Miller et al. (1992) BioTechniques 7: 980-990). Preferably, the viral vector is replication deficient (non-autonomous) and is therefore not infectious in the target cell. Preferably, the replication deficient virus is a minimal virus that retains only the genomic sequence necessary to encapsidate its genome to produce a viral particle. Most preferred are defective viruses lacking viral genes completely or nearly completely. The use of defective viral vectors allows administration to cells in specific local areas without the concern that the vector can infect other cells, thereby allowing tissue specific targeting. For example, Kanno et al. (1999) Cancer Gen. Ther. 6: 147-154; Kaplitt et al. (1997) J. MoI. Neurosci. Meth. 71: 125-132; and Kaplitt et al. (1994) J. MoI. Neuro-Onc. 19: 137-142.

  Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver the nucleic acids of the invention to various cell debris. Attenuated adenoviral vectors, such as those described by Stratford-Perricaudet et al. (1992) (J. Clin. Invest. 90: 626-630) are desirable in some instances. Various replication-defective adenoviruses and minimal adenoviral vectors have been reported (PCT publications WO94 / 26914, WO94 / 28938, WO94 / 28152, WO94 / 12649, WO95 / 02697, and WO96 / 22378). The replication-deficient recombinant adenovirus of the present invention can be produced by any technique known in the art (Leverro et al. (1991) Gene 101: 195; EP185573; Graham (1984) EMBO J. 3: 2917; Graham et al. (1977). J. Gen. Virol. 36:59).

  Adeno-associated virus (AAV) is a relatively small DNA virus that can be integrated into the genome of the cells to which it infects in a stable site-specific manner. They can infect a broad spectrum of cells without inducing any effect on cell growth, morphology or differentiation, and they are believed not to be involved in human pathology. The use of AAV-derived vectors to transfer genes in vitro and in vivo has been reported (eg, Donsante et al. (2001) Gene Ther. 8: 1343-1346; Larson et al. (2001) Adv. Exp. Med. Bio. 489). 45-57; PCT publications WO 91/18088 and WO 93/09239; U.S. Patents 4,797,368 and 5,139,941; and EP 488528 B1).

  In another embodiment, the gene is, for example, US Pat. Nos. 5,399,346, 4,650,764, 4,980,289, and 5,124,263; Mann et al. (1983) Cell 33: 153; Markowitz et al. (1988). ) J. et al. Virol. 62: 1120; can be introduced into retroviral vectors as described in EP453242 and EP178220. Retroviruses are integrated viruses that infect dividing cells.

Lentiviral vectors can be used as agents for direct delivery and sustained expression of nucleic acids encoding antibody molecules of the invention in several tissue types, such as brain, retina, muscle, liver and blood. Vectors can efficiently transduce dividing and non-dividing cells in these tissues and maintain long-term expression of antibody molecules. For a discussion, see Zuffery et al. (1998) J. MoI. Virol. 72: 9873-80 and Kafri et al. (2001) Curr. Opin. Mol. Ther. 3: 316-326. Lentiviral packaging cell lines are available in the art and are generally known to facilitate the production of high titer lentiviral vectors for gene therapy. Examples include the tetracycline-inducible VSV-G pseudotyped lentiviral packaging cell line capable of generating viral particles at a value higher than 10 ⁶ IU / ml for at least 3-4 days, see Kafri et al. Virol. 73: 576-548. Vectors produced by inducible cell lines can be concentrated as needed to efficiently transduce non-dividing cells in vitro and in vivo.

  Sindbis virus is a member of the genus Alphavirus that has been extensively studied since its discovery in various regions of the world in 1953. Gene transduction based on alphaviruses, particularly Sindbis virus, has been well studied in vitro (Straus et al. (1994) Microbiol. Rev. 58: 491-562; Bredenbeek et al. (1993) J. Virol. 67; 6439. -6446; see Iijima et al. (1999) Int. J. Cancer 80: 110-118; and Sawai et al. (1998) Biochim. Biophys. Res. Comm. 248: 315-323). Alphavirus vectors are based on many properties such as rapid manipulation of expression constructs, production of high titer storage solutions of infectious particles, infection of non-dividing cells, and high levels of expression, leading to the induction of other viruses to be developed. It is a desirable alternative to vector systems (Strauss et al. (1994) Microbiol. Rev. 58: 491-562). Sindbis virus for gene therapy has also been reported. (Wahlfors et al. (2000) Gene. Ther. 7: 472-480 and Lundstrom (1999) J. Recep. Sig. Transduc. Res. 19 (1-4): 673-686).

  In another embodiment, the vector can be introduced into cells by lipofection or using other transfection facilitating agents (peptides, polymers, etc.). Synthetic cationic lipids can be used to produce liposomes for in vivo and in vitro transfection of genes encoding markers (Felner et al. (1987) Proc. Nat'l. Acad. Sci. USA 84: 7413-7417 and Wang et al. (1987) Proc. Nat'l. Acad. Sci. USA 84: 7851-7855). Useful lipid compounds and compositions for transfer of nucleic acids are described in PCT Publications WO 95/18863 and WO 96/17823 and US Pat. No. 5,459,127.

  It is also possible to introduce the vector in vivo as a naked DNA plasmid. Naked DNA vectors for gene therapy can be obtained in any desired host cell by methods known in the art, such as electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. (See, eg, Wilson et al. (1992) J. Biol. Chem. 267: 963-967; Williams et al. (1991) Proc. Nat'l. Acad. Sci. USA 88: 2726-2730). Receptor-mediated DNA delivery approaches can also be used (Wu et al. (1988) J. Biol. Chem. 263: 14621-14624). US Pat. Nos. 5,580,859 and 5,589,466 disclose the delivery of exogenous DNA sequences without the use of transfection facilitating agents in mammals. A relatively low voltage, high efficiency in vivo DNA transfer technique called electrotransfer has also been reported (Vilquin et al. (2001) Gene Ther. 8: 1097; Payen et al. (2001) Exp. Hematol. 29: 295. -300; Mir (2001) Bioelectrochemistry 53: 1-10; PCT publications WO99 / 01157, WO99 / 01158 and WO99 / 01175).

  The gene therapy methods outlined herein may be performed in vivo, or they may be performed ex vivo, in which case cells are removed from the subject, transformed with in vitro gene therapy methods, and then the subject. Reintroduced in. See, for example, Wollall (2005) Pediatr. Nephrol. 20 (2): 118-24.

(Example 14)
Cassette mutagenesis of the parent antibody CDRs Optimization of the CDR sequences of anti-human IL-17A antibodies of the present invention (eg 16C10) is performed using shotgun scanning mutagenesis. Alanine scanning mutagenesis is used to determine which residues in the CDR are most important for IL-17A binding (see Example 19). Replacing the codon for one or more internal residues of the CDR with one or more alanine codons or replacing the alanine codon with glycine codons and treating the resulting antibody (eg, various other examples herein) IL-17A binding affinity, as shown in IC50 for receptor blocking in competition tests, bioassay). Codon substitutions are known in the art including, but not limited to, site-directed mutagenesis (eg Kunkel, Proc. Nat'l. Acad. Sci. USA (1985) 82: 488) and PCR mutagenesis. Any method may be used. Residues important for IL-17A binding may also be determined by examining the structure of the IL-17A antibody complex, eg, the X-ray crystal structure. Antibody CDR residues that are within the contact distance of IL-17A or that are substantially buried in the formation of the IL-17A antibody complex are candidates for further optimization.

  The residues with the greatest sensitivity to the mutation are then further examined, for example by homologous scanning mutagenesis. In this example, conservative amino acid substitutions with homologous amino acids are performed on target residues to search for antibodies having excellent quality. Non-conservative mutations are also possible, although it may disrupt IL-17A binding simultaneously.

  Alternatively, advanced antibody sequences may be formed using affinity mutations, in which case selected residues in the CDR are mutated to result in all possible amino acid substitutions at that position. . In another embodiment, as described in WO 2005/044853, fewer than all 20 possible natural amino acids are used for substitution, thereby reducing the number of potential sequences to a more manageable level. While reducing, chemical diversity at each position is still provided using a limited number of amino acids that are chosen to be optimally diverse (eg, typical hydrophobic, uncharged polarities). Of basic and acidic amino acids). Such affinity maturation is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 when including non-standard or modified amino acids. , 17, 18, 19, 20, or more, by substitution at the position of interest with any quantity of amino acids.

(Example 15)
Cross-reactivity with human tissue The tendency of the humanized anti-human IL-17A antibody hu16C10 to cross-reactivity with non-target tissues in human subjects was tested as follows. Hu16C10 was exposed to tissue samples after forming a precomplex by preincubating with a biotinylated secondary antibody. The antibody complex (20 μg / ml antibody) was then mixed with tissue or other sample and allowed to bind by incubating. The bound secondary antibody was then detected using ABC immunoperoxidase detection (Vector Labs, Burlingame, California, USA). Tuson et al. (1990) J. MoI. Histochem Cytochem. 38 (7): 923-6. A sample with an unrelated human IgG1 antibody was used as a negative control.

  Immunohistochemistry (IHC) staining was performed on rhIL-17A protein spots on UV resin slides (Adhesive Coated Slides, Instruments, Inc., St. Louis, Missouri, USA), liver of mice infected with adenovirus encoding IL-17A. It was performed with hu16C10 on several positive control target tissues including cells and human rheumatoid arthritis tissues. IHC revealed binding in all three positive controls (+++).

  Next, cross-reactivity was examined by performing IHC on a panel of human tissues (total 32 specimens). All these human tissue samples were mounted on UV resin slides. Samples were obtained from 3 donors for each tissue. Human tissues screened include adrenal gland, bladder, cerebellum, cerebral cortex, colon, faropian tube, myocardium, kidney, liver, lung, lymph node, mammary gland, ovary, pancreas, parathyroid, pituitary gland, placenta, prostate, retina Skeletal muscle, skin, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, ureter, uterus, and cervix (uterus). IHC was negative in all 32 tissues.

  This lack of cross-reactivity has several potential advantages in the therapeutic use of the anti-IL-17A antibodies of the present invention, such that the antibody is due to non-specific binding to other tissues. Reducing loss (the resulting reduction in therapeutic effect) and reducing the potential for adverse effects associated with unwanted tissue binding.

(Example 16)
Treatment of collagen-induced arthritis with anti-IL-17A antibodies Collagen-induced arthritis (CIA) is a widely accepted mouse model for rheumatoid arthritis in humans. Anti-IL-17A therapy to treat rheumatoid arthritis by administering anti-IL-17A antibody 1D10 of the present invention (parent rat antibody, not its humanized form) to mice expressing CIA Assess your ability.

  The operating method was as follows. On day 0, male B10. RIII mice were immunized intradermally at the base of the tail with bovine type II collagen emulsified in complete Freund's adjuvant. On day 21, mice were challenged intradermally with bovine type II collagen emulsified in incomplete Freund's adjuvant delivered at the base of the tail. When the first sign of severe arthritis in the immunized group occurred (after day 21), all remaining immunized mice were randomly assigned to the various dose groups. Animals were treated with either 800 μg, 200 μg, or 50 μg of anti-IL-17A antibody 1D10; 200 μg isotype control antibody; or diluent. Treatment was performed subcutaneously on the first day of disease onset in immunized mice, and thereafter once a week for a further 4 times. Mice were sacrificed on day 35 and limbs were fixed in 10% neutral buffered formalin and subjected to tissue processing and sectioning. The limbs were analyzed by pathologists for the following histopathological parameters: reactive synovium, inflammation, pannus formation, cartilage destruction, bone erosion, and bone formation. Each parameter was graded using the following disease scale: 0 = no disease, 1 = minimal, 2 = mild, 3 = moderate, 4 = severe. Furthermore, the limbs are evaluated using a visual disease severity score (DSS), which measures edema and erythema on a scale of 0 to 3, where 0 is a normal limb and 1 is a limb. It is assumed that one middle finger is inflamed, 2 is inflamed in 2 fingers or palms of its limbs, and 3 is inflamed in palms and fingers of limbs. A score of 2 and 3 is referred to herein as a limb with severe or severe inflammation.

  The results are shown in FIGS. Each data point represents one limb rather than the average of all four limbs for an animal or across all animals. The reduction in the number of limbs showing high pathological scores is due to the three scales of pathology (visual DSS, limb edema and erythema, cartilage) when anti-IL-17A1D10 concentrations (28 and 7 mg / kg) higher than the test concentration are used. Injury and bone erosion) were statistically significant. Results with the lowest concentration (2 mg / kg) were statistically significant for bone erosion and reduced for visual DSS and cartilage damage. Similar benefits were observed in reducing the production of cartilage destructive enzymes (matrix metalloproteases MMP-2, MMP-3, MMP-13) in the inflamed limb.

  However, visual assessment of limb inflammation may underestimate the therapeutic benefits of anti-IL-17A treatment of CIA mice, such as reduced bone erosion. In another experiment, highly inflamed limbs (DSS score 2 or 3) from CIA mice were analyzed for bone erosion using histopathology or micro-computed tomography (micro CT). Although this study had an extremely reduced percentage of limbs with high inflammation as anti-IL-17A treated animals (see, eg, FIG. 3A), there still remain many limbs with high inflammation. This was possible because it was possible to compare highly inflammatory limbs (DSS = 2 or 3) in all dose groups including antibody-free controls. FIG. 3D shows a plot of bone erosion for a highly inflamed limb obtained from diluent, isotype control (rIgG1) and anti-IL-17A antibody treated animals. Histopathologically measured bone erosion was significantly reduced in the limbs of anti-IL-17A treated animals compared to antibody-free controls, despite similar DSS scores. The results show that reduced bone erosion is achieved with anti-IL-17A treatment even in limbs where there was no apparent improvement in inflammation as measured by DSS score.

  Similar results have been observed when using micro CT to measure bone mineral density (BMD) for joints in highly inflamed limbs in CIA mice. Table 15 shows BMD of limbs with a disease severity score of 0 to 3 for CIA animals administered either anti-IL-17A antibody of the invention (1D10) or isotype control (25D2). Even in the case of joints with the same visual disease severity, the 1D10 antibody treated group only had about half the decrease in bone mineral density observed in the isotype control treated animals.

骨侵食の場合と同様、軟骨破壊及びパンヌス形成（炎症組織の過剰な折り畳みを形成する滑膜管壁の増殖）もまた抗ｈＩＬ−１７Ａ（１Ｄ１０）投与ＣＩＡマウスにおいて低減していた。組織病理学的検討によれば、抗ＩＬ−１７Ａ抗体投与は、希釈剤又はアイソタイプ投与対照と比較した場合に、重度の病理学的状態を示す肢の数を低減したのみならず、目視による検査に基づいて等しく炎症を有する外観を呈していた（ＤＳＳスコア２又は３）肢における病理学的状態をも低減したことを示している。

As with bone erosion, cartilage destruction and pannus formation (proliferation of the synovial tract wall forming excessive folding of inflamed tissue) was also reduced in anti-hIL-17A (1D10) treated CIA mice. According to histopathological examination, administration of anti-IL-17A antibody not only reduced the number of limbs exhibiting a severe pathological condition when compared to diluent or isotype administration controls, but also visual inspection. It also shows that the pathological condition in the limbs that had an equally inflamed appearance based on (DSS score 2 or 3) was also reduced.

  The observation that treatment with anti-IL-17A antibody significantly reduced bone erosion in the CIA model of joint inflammation prevented one of the most debilitating and irreversible effects of RA in humans It suggests that you will find it useful. Furthermore, the observation that bone erosion has been reduced even in highly inflamed limbs, the simple visual assessment of joint inflammation accurately measures treatment effects in the laboratory or ultimately in the hospital. Suggests that you may not. Measurement of bone erosion is necessary to track the effects of therapeutic treatment. Such methods include, but are not limited to, standard 2-DX ray detection, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound (US), and scintigraphy. G., Guermazi et al. (2004) Semin. Musculoskelet. Radiol. 8 (4): 269-285.

(Example 17)
BAL Neutrophil Recruitment Test of Anti-IL-17A Antibody The ability of the anti-IL-17A antibody of the present invention to block IL-17A activity in vivo is evaluated in a bronchoalveolar lavage fluid (BAL) neutrophil recruitment test. did. Briefly, on day -4, 5-week-old female BALB / cAnN mice (Taconic Farms, Germantown, New York, USA) were treated with anti-IL-17A antibody or isotype control of the present invention at 0, 10 antibodies per mouse. , 30, 40, 60, 100 μg by subcutaneous injection. On day-1 mice were stimulated by intranasal administration of 1 μg rhIL-17A (or PBS alone control) in 50 μl PBS under mild isofuran anesthesia.

On day 0, the level of neutrophils present in the BAL fluid was measured as follows. Mice were euthanized with CO ₂ and blood samples were collected from which the concentration of anti-IL-17A antibody was determined. The needle was inserted into the upper cervical trachea by tracheotomy and BAL fluid was collected by introducing and draining 0.3 ml PBS three times. The BAL fluid was centrifuged (400 × g, 4 ° C. for 10 minutes), and the cell pellet was resuspended in PBS. The total cell number was measured with a hemocytometer using trypan blue solution. Blood cell fractionation was performed on a cytospin preparation by Wright-Giemsa staining (Sigma-Aldrich, St. Louis, Missouri, USA) using an oil immersion microscope (original magnification × 1000) according to standard morphological criteria. It was. Cell counts were performed on 200 or 300 cells (lymphocytes, monocytes, neutrophils, eosinophils) to determine percent BAL neutrophils.

  The results are shown in FIG. Data are shown for three anti-IL-17A antibodies (1D10, 16C10, and 4C3) of the present invention and controls. The percentage of neutrophils in BAL fluid as a percentage of total leukocytes for individual laboratory animals was plotted as a function of serum antibody concentration, with the left segment (0 to 1) on the horizontal axis as a control, as indicated in the legend. . The control shows that rhIL-17A stimulation induces significant neutrophil recruitment that is not reduced by administration of an isotype control antibody (administered at the same level as the anti-IL-17A antibody). In contrast, anti-IL-17A data shows a dose-dependent reduction in rhIL-17A-induced neutrophil recruitment, with neutrophil recruitment at serum antibody concentrations above 40-60 μg / ml. Essentially blocked.

(Example 18)
Treatment of Rheumatoid Arthritis (RA) with Anti-IL-17A Antibody A human subject diagnosed with RA who had an inadequate response to one or more DMARDs is a humanized anti-IL-17A antibody of the present invention. Selected for treatment with Subjects are maintained with methotrexate (10 mg / week) and optionally treated with prednisone for 2 weeks. Subjects receive 50 or 100 mg of anti-IL-17A antibody monthly by subcutaneous administration. Doses are adjusted for a particular subject according to standard clinical criteria and based on clinical response.

  Response to therapy is assessed by measuring ACR scores based on criteria developed by the American College of Rheumatology. ACR score is a reduction in the number of swollen and tender joints, patient overall assessment, physician overall assessment, pain scale, self-assessed disorders, and acute phase reactants (erythrocyte sedimentation rate or C-reactive protein) A composite score that integrates a number of clinical parameters such as See Felson et al. (1995) Arthritis & Rheumatology 38; 727-735. Subjects are considered improved if they show a score of ACR 20 or higher at 24 weeks of treatment. Furthermore, the humanized anti-IL-17A antibody of the present invention is compared by comparing the administration group with the placebo group in clinical trials using populations of subjects who have achieved various ACR scores (for example, ACR20, ACR50 and ACR70). Clinical efficacy can be evaluated.

(Example 19)
Epitope determination The amino acid residues that are important for binding of the antibodies of the invention, for example rat 16C10, were determined as follows.

  The first set of experiments shows that rat antibody 16C10 can bind to human IL-17A (hIL-17A) but not to mouse IL-17A (mIL-17A) or a related cytokine, human IL-17F. Based on observation results. Each of these three proteins was linked to an N-terminal FLAG® peptide tag (see residues 1-9 of SEQ ID NO: 42). In order to identify amino acid residues important for 16C10 binding, various peptide subsequences of FLAG-tagged hIL-17A, mIL-17A and IL-17F are mixed with restriction fragments of the relevant gene. To form a hybrid polypeptide. By determining the binding of these hybrid polypeptides to anti-16C10 in the Amplified Luminescent Proximity Homogenous Assay (AlphaScreen, Packard BioScience, Wellesley, Massachusetts, USA), which segment is the hIL-17 . Biotinylated antibody 16C10 was conjugated to streptavidin donor beads and hybrid polypeptide was conjugated to acceptor beads with anti-FLAG® antibody (Packard BioScience). Donor and acceptor beads were mixed, illuminated at 680 nm, and emission was measured at 520-620 nm. Since the acceptor bead is held in the vicinity of the donor bead and emits light when singlet oxygen diffuses from the excited donor bead to the acceptor bead, the hybrid polypeptide bound to antibody 16C10 Binding was measured as enhanced fluorescence in samples containing.

  The results show that anti-16C10 binds to hybrid polypeptides containing amino acid residues 50-132, 63-132, 1-87, 1-112 and 63-87 of hIL-17A, which is important for 16C10 binding It has been revealed that these residues are present at residues 63-87 of hIL-17A (PSVIWEAKCRHLGCINADG NVDYHM). The numbering of all residues in this example refers to the sequence of hIL-17A (SEQ ID NO: 40). For example, mIL-17A and IL-17F polypeptides substituted with residues 63-87 of hIL-17A were able to bind antibody 16C10, but not intact mIL-17A and IL-17F.

  Point mutations were also examined to determine which amino acid residues are important for antibody 16C10 binding by introduction into hIL-17A. In one experiment, several residues (45, 46, 51, 52, 54, 55, 56, 57, 58, 60, 61, 62, 67, 68, 70, 72, 73, 78, 80, 82, 84, 85, 86, 88, 93, 94, 95, 100, 101, 102, 105, 108, 110, 111, 113, 114) in which an alanine codon is introduced instead of the native amino acid. Mutagenesis was performed. A mammalian expression plasmid carrying a gene encoding a mutant of hIL-17A was transiently transfected into human embryonic kidney (HEK) 293 cells. Supernatants were analyzed by AlphaScreen as described above for FLAG® peptide tag quantification and for anti-16C10 binding. None of the single amino acid substitutions significantly reduced antibody 16C10 binding. Other point mutations were made in which human IL-17F or mouse IL-17A residues were replaced at various positions within the 63-87 fragment, ie L74Q, G75R, V83E, Y85H. None of these individual point mutations suppressed antibody 16C10 binding, but hIL-17A with all four changes showed substantially reduced binding, in the 63-87 fragment of hIL-17A. Residues, and more particularly, the residue in the 74-85 fragment (LGCINADGNVDY) was confirmed to be important for 16C10 binding.

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Citation of the above publications or documents does not imply any prior art admission, nor constitutes any admission as to the content or date of these publications or documents. All references cited herein are hereby incorporated by reference to the same extent as if each individual publication, patent application, or patent was specifically and individually incorporated herein by reference. It is.

Claims (45)

At least one antibody light chain variable region having at least a specified number of CDR sequences selected from the group consisting of SEQ ID NOs: 11-13 or a binding fragment thereof; and
At least one antibody heavy chain variable region having at least a particular number of CDR sequences selected from the group consisting of SEQ ID NOs: 14-20, or a binding fragment thereof,
A binding compound that binds to human IL-17A, comprising
Wherein the specific compound is 1.   The binding compound according to claim 1, wherein the specific number is 2.   The binding compound according to claim 1, wherein the specific number is 3. The light chain variable region comprises the CDR sequences of SEQ ID NOs: 11-13;
4. The binding compound of claim 3, wherein the heavy chain variable region comprises the CDR sequences of SEQ ID NOs: 14, 17 and 20. The light chain variable region comprises the CDR sequences of SEQ ID NOs: 11-13;
4. The binding compound of claim 3, wherein the heavy chain variable region comprises the CDR sequences of SEQ ID NOs: 14, 16, and 19.   4. The light chain variable region comprises the sequence of SEQ ID NO: 5 having up to 10 conservative substitutions, and the heavy chain variable region comprises the sequence of SEQ ID NO: 6 having up to 10 conservative amino acid substitutions. Binding compound. The binding compound is
A light chain variable region comprising SEQ ID NO: 5, and
A heavy chain variable region comprising SEQ ID NO: 6,
The binding compound according to claim 6, which is an antibody comprising   The light chain consists essentially of amino acids 1-220 of SEQ ID NO: 2 containing up to 10 conservative substitutions, and the heavy chain consists essentially of amino acids 1-454 of SEQ ID NO: 4 containing up to 10 conservative substitutions The binding compound according to claim 3, wherein   9. The binding compound of claim 8, wherein the light chain consists essentially of amino acids 1-220 of SEQ ID NO: 2 and the heavy chain consists essentially of amino acids 1-454 of SEQ ID NO: 4.   10. The binding compound of claim 9, wherein the light chain consists of amino acids 1-220 of SEQ ID NO: 2 and the heavy chain consists essentially of amino acids 1-454 of SEQ ID NO: 4.   A binding compound that binds to human IL-17A comprising a light chain variable region having at least 90% homology to SEQ ID NO: 5 and a heavy chain variable region having at least 90% homology to SEQ ID NO: 6.   4. An isolated nucleic acid encoding at least one of a light chain variable region and a heavy chain variable region of the binding compound of claim 3.   13. The nucleic acid of claim 12, wherein the amino acid sequence of the light chain variable region is SEQ ID NO: 5 and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 6.   The nucleic acid according to claim 13, comprising the sequence of SEQ ID NOs: 1 and 3, or the sequences of SEQ ID NOs: 62 and 63.   15. An expression vector comprising the nucleic acid of claim 14, wherein the nucleic acid is operably linked to a regulatory sequence that is recognized by the host cell when the host cell is transfected with the vector. .   16. The expression vector of claim 15, wherein the expression vector has ATCC accession number PTA-7675.   A host cell comprising the vector of claim 16. Producing a polypeptide comprising light and heavy chain variable regions by culturing the host cell of claim 17 in a medium under conditions in which the nucleic acid sequence is expressed; and
Recovering the polypeptide from the host cell or medium;
A method for producing a polypeptide comprising:   The anti-human IL-17 antibody hu16C10 whose antibody sequence is encoded by the expression vector of claim 16.   An antibody capable of cross-blocking the binding of the binding compound of claim 10 to human IL-17A in a cross-blocking test.   20. An antibody that binds to the same epitope on human IL-17A as the epitope bound by antibody 16C10 of claim 19.   An antibody that binds to human IL-17A at an epitope comprising amino acid residues 74-85 of the human IL-17A protein sequence of SEQ ID NO: 40. A light chain variable region comprising the CDR sequences of SEQ ID NOs: 26-28, and
A heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 29-31,
A binding compound that binds to human IL-17A.   24. The binding compound of claim 23, wherein the light chain variable region comprises the sequence of SEQ ID NO: 22 and the heavy chain variable region comprises the sequence of SEQ ID NO: 23.   The binding compound according to claim 23, wherein the binding compound is a monoclonal antibody. (A) a light chain variable region comprising the CDR sequences of SEQ ID NOs: 48-50 and a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 51-53, or
(B) a light chain variable region comprising the CDR sequences of SEQ ID NOs: 56-58, and a heavy chain variable region comprising the CDR sequences of SEQ ID NOs: 59-61,
A binding compound that binds to human IL-17A.   27. The binding compound of claim 26, wherein the binding compound is a humanized monoclonal antibody. The binding compound further has the following properties:
a) the binding compound binds to human IL-17A with an equilibrium dissociation constant (K _d ) of 100 pM or less,
b) the binding compound has an IC ₅₀ of 5 nM or less in a test for suppression of IL-6 production in human IL-17A stimulated normal human dermal fibroblasts, wherein the human IL-17A stimulation is 1 nM human IL- At 17A,
c) The binding compound has an IC ₅₀ of ₅₀₀ pM or less in an in vitro test of the biological activity of human IL-17A, wherein the in vitro test measures the biological activity of 100 pM of human IL-17A ,
d) Reduce IL-17A-induced neutrophil recruitment to the lung by 50% or more (measured as percent of BAL neutrophils) when the binding compound is administered to mice at a serum concentration of 50 μg / ml. Can
e) The binding compound binds to cynomolgus IL-17A with an affinity of 20 times or less of the affinity (K _d ) for human IL-17A, where the binding compound also inhibits the activity of cynomolgus IL-17A And
f) the binding compound binds human IL-17A with an affinity at least 1000 times its affinity (K _d ) for mouse or rat IL-17A;
A binding compound that binds to human IL-17A, having at least one of the following: The binding compound has the following properties:
a) the binding compound binds to human IL-17A with an equilibrium dissociation constant (K _d ) of 20 pM or less,
b) The binding compound has an IC ₅₀ of 1 nM or less in a test for inhibition of IL-6 production in human IL-17A stimulated normal human dermal fibroblasts, wherein the human IL-17A stimulation is 1 nM human IL- At 17A, and
c) said binding compound has the following _{IC 50} 100pM in vitro assay of the biological activity of human IL-17A, where the test of the in vitro to measure the biological activity of 100pM of human IL-17A ,
29. A binding compound according to claim 28 having at least one of   29. The binding compound of claim 28, wherein the binding compound is a humanized or fully human monoclonal antibody.   A method of treating a subject comprising administering to a human subject in need of treatment a therapeutically effective amount of a binding compound that binds to human IL-17A and neutralizes the activity of human IL-17A, comprising: The binding compound comprises an antibody light chain variable region and an antibody heavy chain variable region, wherein the light chain variable region comprises SEQ ID NO: 5 and the heavy chain variable region comprises SEQ ID NO: 6.   32. The method of claim 31, wherein the binding compound is a monoclonal antibody.   35. The method of claim 32, wherein the human subject has an inflammatory or autoimmune disorder.   34. The method of claim 33, wherein the human subject has rheumatoid arthritis.   34. The method of claim 33, wherein the human subject has inflammatory bowel disease.   32. The method of claim 31, further comprising administering another immunosuppressive or anti-inflammatory agent. A binding compound that binds to human IL-17A and neutralizes human IL-17A activity, wherein the binding compound comprises an antibody light chain variable region and an antibody heavy chain variable region, wherein the light chain variable region comprises A binding compound comprising SEQ ID NO: 5 and the heavy chain variable region comprises SEQ ID NO: 6, and
A pharmaceutically acceptable carrier or diluent,
A composition comprising   38. The composition of claim 37, further comprising administering another immunosuppressive or anti-inflammatory agent.   6. The binding compound of claim 5, further comprising a heavy chain constant region, wherein the heavy chain constant region comprises a γ1, γ2, γ3, or γ4 human heavy chain constant region or a variant thereof.   40. The binding compound of claim 39, comprising a γ1 human heavy chain constant region or a variant thereof.   40. The binding compound of claim 39, comprising a γ4 human heavy chain constant region or a variant thereof.   6. The binding compound of claim 5, further comprising a light chain constant region, wherein the light chain constant region comprises a kappa human light chain constant region. 6. The binding compound according to claim 5, wherein the binding compound is an antibody fragment selected from the group consisting of Fab, Fab ′, Fab′-SH, Fv, scFv, F (ab ′) ₂ , and diabody. The binding compound according to claim 6, wherein the binding compound is an antibody fragment selected from the group consisting of Fab, Fab ', Fab'-SH, Fv, scFv, F (ab') ₂ , and diabody.   29. A binding compound according to any of claims 1, 11, 23, 26 or 28 that inhibits human IL-17A activity.

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